SCF Extract Containing Cardiac Glycoside

ABSTRACT

A supercritical fluid (SCF) extract of a cardiac glycoside-containing plant mass is provided. The extract can be included in a pharmaceutical composition containing an extract-solubilizing amount of solubilizer. Oleandrin is included within the extract when a cardiac glycoside-containing plant, such as  Nerium oleander , is extracted by SCF extraction. The extract can also contain one or more other SCF extractable pharmacologically active agents. The composition can be used to treat a wide range of disorders that are therapeutically responsive to a cardiac glycoside.

CROSS-REFERENCE TO EARLIER FILED APPLICATION

This application claims the priority of and is a continuation in part ofPCT International Application No. PCT/US06/29061 filed Jul. 26, 2006,which claims the priority of and is a continuation in part of U.S.application Ser. No. 11/191,650 filed Jul. 28, 2005, the entiredisclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns a pharmaceutical formulation comprisingoleandrin present as a supercritical fluid (SCF) extract comprising acardiac glycoside. The formulation comprises one or more solubilizers,such as surfactants, that aid in solubilization, dispersion oremulsification of at least the cardiac glycoside, and optionally otherpharmacologically active components of the extract, in the formulationwhen the formulation is placed in an aqueous environment. The inventionalso concerns SCF extracts comprising cardiac glycoside and methods ofuse thereof for the treatment of cardiac glycoside-responsive diseasesor disorders.

BACKGROUND OF THE INVENTION

Nerium oleander is an ornamental plant widely distributed in subtropicalAsia, the southwestern United States, and the Mediterranean. Its medicaland toxicological properties have long been recognized. It has beenused, for example, in the treatment of hemorrhoids, ulcers, leprosy,snake bites, and even in the induction of abortion. Oleandrin, animportant component of oleander extract, is a potent inhibitor of humantumor cell growth (Afaq F et al. Toxicol. Appl. Pharmacol. 195:361-369,2004). Oleandrin-mediated cell death is associated with calcium influx,release of cytochrome C from mitochondria, proteolytic processes ofcaspases 8 and 3, poly(ADP-ribose) polymerase cleavage, and DNAfragmentation.

It has been demonstrated that oleandrin is the principal cytotoxiccomponent of Nerium oleander (Newman, et al., J. Herbal Pharmacotherapy,vol. 13, pp. 1-15, 2001). Oleandrin is a cardiac glycoside that isexogeneous and not normally present in the body. Oleandrin inducesapoptosis in human but not in murine tumor cell lines (Pathak et al.,Anti-Cancer Drugs, vol. 11, pp. 455-463, 2000), inhibits activation ofNF-kB (Manna et al., Cancer Res., vol. 60, pp. 3838-3847, 2000), andmediates cell death in part through a calcium-mediated release ofcytochrome C (McConkey et al., Cancer Res., vol. 60, pp. 3807-3812,2000). A Phase I trial of a hot water oleander extract has beencompleted recently (Mekhail et al., Am. Soc. Clin. Oncol., vol. 20, p.82b, 2001). It was concluded that oleander extracts can be safelyadministered at doses up to 1.2 ml/m²/d. No dose limiting toxicitieswere found.

In addition to being selectively cytotoxic for tumor cells, cardiacglycosides may also enhance cell response to cytotoxic actions ofionizing radiation. Ouabain, a cardiac glycoside endogeneous to thebody, was reported to enhance in vitro radiosensitivity of A549 humanlung adenocarcinoma cells but was ineffective in modifying theradioresponse of normal human lung fibroblasts (Lawrence, Int. J.Radiat. Oncol. Biol. Phys., vol. 15, pp. 953-958, 1988). Ouabain wassubsequently shown to radiosensitize human tumor cells of differenthistology types including squamous cell carcinoma and melanoma(Verheye-Dua et al., Strahlenther. Onkol., vol. 176, pp. 186-191, 2000).Although the mechanisms of ouabain-induced radiosensitization are stillnot fully explained, inhibition of repair from sublethal radiationdamage and an increase in radiation-induced apoptosis have been advancedas possibilities (Lawrence, 2000; Verheye Dua et al., 2000; Verheye-Duaet al., Strahlenther. Onkol., vol. 172, pp. 156-161, 1996). The cardiacglycoside oleandrin also has the ability to enhance the sensitivity ofcells to the cytotoxic action of ionizing radiation. See U.S. patentapplication Ser. No. 10/957,875 to Newman, et al. and Nasu et al.,Cancer Lett. Vol 185, pp. 145-151, 2002).

Chen et al. (Breast Cancer Research and Treatment (2006), 96, 1-15)suggest that cardiac glycosides, such as ouabain and digitalis, might beuseful toward developing anti-breast cancer drugs as both Na⁺, K⁺-ATPaseinhibitors and ER antagonists.

Smith et al. (Biochemical Pharmacology (2000), 62, 1-4) report thatANVIRZEL, and its key cardiac glycoside component oleandrin, inhibitsthe exportation of fibroblast growth factor-2 (FGF-2) from the prostatecancer cell lines PC3 and DU145.

Newman et al. (J. Experimental Therapeutics and Oncology (2006), 5,167-181) report that incubation of human malignant melanoma BRO cellswith oleandrin results in a time-dependent formation of reactive oxygenspecies, superoxide anion radicals, that mediate mitochondrial injuryand loss of cellular GSH pools.

U.S. Pregrant Patent Application Publication No. 20050112059 to Newmanet al. discloses the enhancement of radiotherapy in the treatment ofcancer by administration of oleandrin.

Extraction of glycosides from plants of Nerium species has traditionallybeen carried out using boiling water. The process of using boiling wateras an extraction method to obtain active ingredients from Neriumoleander yields many products. Among these are oleandrin, nerine, andother cardiac glycoside compounds. The plant extracts are useful in thetreatment of cell-proliferative diseases in animals.

Oleandrin extracts obtained by hot-water extraction of Nerium oleander,sold under the trademark ANVIRZEL™, are commercially available andcontain the concentrated form or powdered form of a hot-water extract ofNerium oleander. The extract is prepared according to the processdeveloped by Dr. Huseyin Ziya Ozel. U.S. Pat. No. 5,135,745 describes aprocedure for the preparation of the extract of the plant in water. Theextraction of the plant Nerium oleander involves slicing the leaves,cooking the sliced leaves and stems of the plant in water for 2-3 hoursand filtering off the residues. The mixture is heated again. The aqueousextract reportedly contains several polysaccharides with molecularweights varying from 2 KD to 30 KD, oleandrin and oleandrigenin,odoroside and neritaloside. The polysaccharides reportedly includeacidic homopolygalacturonans or arabinogalaturonans. Ozel (ibid, andPlanta Med. 56 (1990), 668) incorrectly reports that the cardiacglycosides are not responsible for the antitumor activity of the aqueousextract.

Muller et al. (Pharmazie. (1991) September. 46(9), 657-663) disclose theresults regarding the analysis of a water extract of Nerium oleander.They report that the polysaccharide present is primarily galacturonicacid. Other saccharides include rhamnose, arabinose and galactose.Polysaccharide content and individual sugar composition ofpolysaccharides within the hot water extract of Nerium oleander havealso been reported by Newman et al. (J. Herbal Pharmacotherapy, (2001)vol 1, pp. 1-16).

U.S. Pat. No. 5,869,060 to Selvaraj et al. pertains to extracts ofNerium species and methods of production. To prepare the extract, plantmaterial is placed in water and boiled. The crude extract is thenseparated from the plant matter and sterilized by filtration. Theresultant extract can then be lyophilized to produce a powder.

U.S. Pat. No. 6,565,897 (U.S. Pregrant Publication No. 20020114852 andPCT International Publication No. WO 2000/016793 to Selvaraj et al.)discloses a hot-water extraction process for the preparation of asubstantially sterile extract.

Erdemoglu et al. (J. Ethnopharmacol. (2003) Nov. 89(1), 123-129)discloses results for the comparison of aqueous and ethanolic extractsof plants, including Nerium oleander, based upon their anti-nociceptiveand anti-inflammatory activities.

Organic solvent extracts of Nerium oleander are also disclosed by Adomeet al. (Afr. Health Sci. (2003) Aug. 3(2), 77-86; ethanolic extract),el-Shazly et al. (J. Egypt Soc. Parasitol. (1996), Aug. 26(2), 461-473;ethanolic extract), Begum et al. (Phytochemistry (1999) Feb. 50(3),435-438; methanolic extract), Zia et al. (J. Ethnolpharmacol. (1995)Nov. 49(1), 33-39; methanolic extract), and Vlasenko et al. (Farmatsiia.(1972) September-October 21(5), 46-47; alcoholic extract).

Supercritical fluid extraction involves the use of a supercritical fluidto selectively extract a particular compound. A supercritical fluid is aliquid or a gas at atmospheric conditions, but becomes supercriticalwhen it is compressed above its critical pressure and heated above itscritical temperature. Supercritical fluids have increased dissolvingpower in their supercritical regions. A supercritical fluid exhibitsproperties between those of a gas and a liquid, and has the capacity todissolve compounds that may only dissolve poorly or not at all in thegas or liquid state. Supercritical fluids are ideal for extraction ofthese compounds because they have high dissolving power at highdensities and demonstrate good fractionation and separation of thecompound from the fluid at lower densities when the pressure ortemperature is changed. The general procedure of using supercriticalcarbon dioxide extraction in food processing industry has been describedby Raventos, et al., in 2002 (M. Raventos, et al., Application andPossibilities of Supercritical CO₂ Extraction in Food ProcessingIndustry: An Overview, Food Sci. Tech. Int. Vol. 8 (5) (2002) 269-284),the entire content of which is hereby incorporated by reference.Raventos et al. report that SCF extraction is not suitable forextraction of polysaccharides from plant material even though hesuggests that a modifier such as ethanol may be used to modify theperformance of an SCF extraction.

U.S. Pregrant Patent Application Publication No. 20040247660 to Singh etal. discloses the preparation of a protein stabilized liposomalformulation of oleandrin for use in the treatment of cancer.

U.S. Pregrant Patent Application Publication No. 20050026849 to Singh etal. discloses a water soluble formulation of oleandrin containing acyclodextrin. The '849 Publication suggests the preparation ofsolid-filled capsules containing the cyclodextrin complex of oleandrin.The oleandrin has been provided as the hot-water extract or the chemicalentity and then treated with the cyclodextrin to form the complex.

U.S. Pregrant Patent Application Publication No. 20040082521 to Singh etal. discloses the preparation of protein stabilized nanoparticleformulations of oleandrin from the hot-water extract. The nanoparticlesare prepared via formation of a liposomal mixture and subsequentevaporation of the organic solvent therein.

Methods to enhance the relative content of oleandrin from plant materialare therefore warranted. While hot water extracts of Nerium oleander mayprovide oleandrin and related cardiac glycosides in relatively lowyield, an improved method for obtaining a concentrated form of cardiacglycosides including oleandrin is needed.

Oleandrin contains a lactone ring that is acid labile and predisposesthe material to acid degradation when orally dosed, so care must betaken in the preparation of liquid formulations to ensure minimizationof acidic species in solution.

None of the known art discloses a pharmaceutical formulation comprisingan extract of Nerium species, in particular, Nerium oleander. None ofthe art discloses or suggests a supercritical fluid extract comprising acardiac glycoside, such as oleandrin. A need remains for more dosageforms that provide suitable delivery of the components of an extract ofNerium species for the treatment of various diseases and disorders. Aneed also remains for improved processes for obtaining cardiacglycosides by extraction from plant material.

SUMMARY OF THE INVENTION

The present invention seeks to overcome some or all of the disadvantagesinherent in the art. The invention provides a supercritical fluid (SCF)extract comprising cardiac glycoside. The extract can be obtained bysupercritical fluid extraction of a cardiac glycoside-containing plantmass. The plant mass can be Nerium species or Thevetia species plantmass. Particular species include Nerium oleander or Thevetia nerifolia.The supercritical fluid extract can comprise at least one otherpharmacologically active agent that contributes to the therapeuticefficacy of the cardiac glycoside when the extract is administered to asubject. It can contribute additively or synergistically to therapeuticefficacy.

The invention also provides a pharmaceutical composition comprising aSCF extract comprising a cardiac glycoside. The cardiac glycoside can bepresent as the primary therapeutic component in the pharmaceuticalcomposition, and one or more other therapeutic components may also bepresent. Some embodiments of the invention include an extract ofoleander plant, such as of Nerium species, e.g. Nerium oleander, or suchas of or of Thevetia species, e.g. Thevetia nerifolia. The extract canbe prepared by supercritical fluid (SCF) carbon dioxide (CO₂) extractionof plant material, such as a dried powder of plant mass, by a processdescribed herein or in a currently-pending U.S. application Ser. No.60/653,210 filed Jul. 28, 2005 in the name of C. Addington and U.S.application Ser. No. 11/191,650 filed Jul. 28, 2005 in the name of C.Addington, the entire disclosures of which are hereby incorporated byreference, or by a process described herein. The SCF extraction can beconducted in the presence of a modifier to enhance extraction of thedesired compound(s) from the plant mass.

Accordingly, the invention also provides a supercritical fluidextraction process of a cardiac glycoside-containing plant mass. Theprocess comprises:

-   treating a cardiac glycoside-containing plant mass with a    supercritical fluid for a period of time sufficient to extract the    cardiac glycoside from the plant mass;-   separating the plant mass from the supercritical fluid; and-   removing the supercritical fluid thereby forming a supercritical    fluid (SCF) extract comprising cardiac glycoside.

The supercritical fluid can further comprise a modifier. The cardiacglycoside can be present as the primary therapeutic component in theextract, and the SCF extract can further comprise at least one otherpharmacologically active agent aside from the cardiac glycoside. Theother active agent may contribute to the therapeutic efficacy of thecardiac glycoside when the extract is administered to a subject. Theother active agent may function additively or synergistically tocontribute to the therapeutic efficacy of the cardiac glycoside. Thecardiac glycoside-containing plant mass can comprise Nerium species orThevetia species. In some embodiments, the other therapeutic agent isnot a polysaccharide, such as an acidic homopolygalacturonan orarabinogalaturonan. In some embodiments, the extract excludes anothertherapeutic agent and/or excludes an acidic homopolygalacturonan orarabinogalaturonan. In some embodiments, the extract comprises one ormore cardiac glycosides and one or more cardiac glycoside precursors(such as cardenolides, cardadienolides and cardatrienolides, all ofwhich are the aglycone constituents of cardiac glycosides, for example,digitoxin, acetyl digitoxins, digitoxigenin, digoxin, acetyl digoxins,digoxigenin, medigoxin, strophanthins, cymarine, ouabain, orstrophanthidin). The extract may further comprise one or more glyconeconstituents of cardiac glycosides (such as glucoside, fructoside,and/or glucuronide) as cardiac glycoside presursors. Accordingly, theextract may comprise one or more cardiac glycosides and two more cardiacglycoside precursors selected from the group consisting of one or moreaglycone constituents, and one or more glycone constituents. Anadvantage of the composition, and dosage form thereof, of the inventionis its ability to provide a solution of the entire, or of at least amajor portion of, extract following oral administration such that all ofthe components are solubilized, emulsified or dispersed when placed inan aqueous environment. Solubilization, dispersion or emulsification canbe the result of simple dissolution, micelle formation orself-emulsification depending upon the combination of excipients used inthe composition. In some embodiments, solubilization of the SCFextraction is not pH dependent. Another advantage is substantiallycomplete dissolution of all of the extract components in apharmaceutical liquid composition comprising the SCF extract.

In some embodiments, the formulation comprises a combination of at leasttwo materials selected from the group consisting of a water soluble(miscible) co-solvent, a water insoluble (immiscible) co-solvent, asurfactant, an antioxidant, a chelating agent, an absorption enhancerand the SCF extract.

One aspect of the invention provides a pharmaceutical compositioncomprising:

-   -   a supercritical fluid extract of oleander plant mass; and    -   an extract-solubilizing amount of at least one solubilizer.

In some embodiments of the invention, the oleander plant mass comprisesNerium species, such as Nerium oleander, or of Thevetia species, such asThevetia nerifolia (otherwise known as yellow oleander). The oleanderplant mass is a cardiac glycoside-containing plant mass. The cardiacglycoside can be oleandrin.

Another aspect of the invention provides a capsule formulationcomprising a capsule shell, and a pharmaceutical composition asdescribed herein. In some embodiments, the capsule formulationcomprises:

-   -   a capsule shell;    -   an oleandrin extract obtained by supercritical fluid extraction        of Nerium species; and    -   an extract-solubilizing amount of at least one solubilizer.

The capsule formulation can be a solid, liquid, or semi-solid. Thesolubilizer can comprise a single component or a mixture of two, three,four, five or more components. Such components may be selected from thegroup consisting of water soluble (miscible) co-solvent, water insoluble(immiscible) co-solvent, surfactant, and antioxidant.

Some embodiments of the invention comprise the SCF extract and:

-   -   at least one water miscible solvent;    -   at least one antioxidant; and    -   at least one surfactant.

The solubilizer is at least a single surfactant, but it can also be acombination of materials such as a combination of: a) surfactant andwater miscible solvent; b) surfactant and water immiscible solvent; c)surfactant, antioxidant; d) surfactant, antioxidant, and water misciblesolvent; e) surfactant, antioxidant, and water immiscible solvent; f)surfactant, water miscible solvent, and water immiscible solvent; or g)surfactant, antioxidant, water miscible solvent, and water immisciblesolvent.

The composition optionally further comprises: a) at least one liquidcarrier; b) at least one emulsifying agent; c) at least one solubilizingagent; d) at least one dispersing agent; e) at least one otherexcipient; or f) a combination thereof.

In some embodiments, water miscible solvent is low molecular weight(less than 6000) PEG, glycol, or alcohol. In some embodiments, thesurfactant is a pegylated surfactant, meaning a surfactant comprising apoly(ethylene glycol) functional group.

Prior to oral administration or exposure to an aqueous solution, someembodiments of the composition are clear, and others are suspensions.Some embodiments of the invention form an emulsion, micellar dispersionor solid dispersion (suspension) in the gastrointestinal (GI) tract of asubject after oral administration or in an aqueous medium.

The dosage form of the invention is adapted for oral administration to asubject and is suitable for the treatment of malignant neoplasticdisease, cancer, tumor, viral infection and other indications, disordersor symptoms that are therapeutically responsive to a cardiac glycoside,such as oleandrin. As used herein, the term “subject” is taken to meanwarm blooded animals such as mammals, for example, cats, dogs, mice,guinea pigs, horses, bovine cows, sheep, and humans. Examples 7-9provide exemplary procedures for the treatment of various disorders withan SCF extract of the invention. Cancer of the rectum, anus, colorectaltissues, head and neck tissues, esophageal tissue, lung (both non smallcell and small cell carcinomas), breast, stomach, pancreas, prostate,liver, kidney, bladder, ureter, ovarian tissue, carcinoid tumors,sarcomas of bone, mesothelioma, and neoplasms of the central nervoussystem can be treated with the SCF extract.

The extract, pharmaceutical composition and pharmaceutical dosage formof the invention can be used to treat a viral infection. The method fordetermining the relative antiviral activity of extracts and cardiacglycosides of the invention is detailed in Example 12. Accordingly, asubject suffering from a viral infection is treated with atherapeutically effective amount of the SCF extract of the inventionthereby providing relief of symptoms associate with or amelioration orprevention (prophylaxis) of the viral infection. A particular viralinfect is HIV infection.

Some embodiments of the liquid composition are anhydrous or have nowater added thereto. The composition may contain endogenous wateralready present in one or more of the components of the composition.Alternatively, the composition contains water added as a separatecomponent thereof.

The invention provides an extract comprising one or more cardiacglycosides, wherein the extract has been prepared by SCF extraction ofcardiac glycoside-containing plant material. In some embodiments, theSCF is carbon dioxide. In some embodiments, the SCF further comprises amodifier (extraction modifier). In some embodiments, the SCF extractfurther comprises at least one non-cardiac glycoside SCF extractablepharmacologically active agent obtained by way of the supercriticalfluid extraction. The non-cardiac glycoside active agent might notcontribute to the therapeutic efficacy of the cardiac glycoside when theextract is administered to a subject in need thereof. Alternatively, atleast one other non-cardiac glycoside supercritical fluid extractablepharmacologically active agent functions additively or synergisticallyto contribute to the therapeutic efficacy of the cardiac glycoside whenthe extract is administered to a subject.

The invention also provides a method of treating a disease or disordertherapeutically responsive to cardiac glycoside therapy in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a supercritical fluid extractcomprising a cardiac glycoside. The invention also provides a method ofinhibiting or reducing the extent of Akt phosphorylation in a cancercell or method of enhancing the expression of pERK in a cancer cell, themethod comprising treating the cell with an effective amount ofsupercritical fluid extract comprising a cardiac glycoside. Theinvention also provides a method of inhibiting the proliferation ofcancer cells comprising treating the cancer cells with an effective ofamount supercritical fluid extract comprising a cardiac glycoside. Themethods can be practiced in vivo or in vitro.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present description and describeexemplary embodiments of the claimed invention. The skilled artisanwill, in light of these figures and the description herein, be able topractice the invention without undue experimentation.

FIGS. 1A and 1B depict comparative HPLC chromatograms for a prior arthot-water extract and an exemplary supercritical fluid extract of theinvention.

FIG. 2 depicts a dissolution profile for an enteric coated liquid-filledcapsule of the invention.

FIG. 3 depicts a photograph of the relevant bands of a gelelectropherogram obtained as part of an assay comparing the activity ofoleandrin, hot-water extract and the SCF extract of the invention towardinhibition of phosphoryation (active form) of Akt and augmentation of(phosphorylation) of Erk.

FIG. 4 is a plot of concentration of oleandrin versus percent growth ofcontrol of PANC-1 cells for two SCF extracts of the invention ascompared to pure oleadrin.

FIG. 5 is a semi-log plot of concentration of oleandrin versus percentgrowth of control of human melanoma BRO cells for an SCF extract of theinvention as compared to a hot water extract.

FIGS. 6A-6D depict comparative HPLC chromatograms for the carbohydrateanalysis of a carbohydrate reference (FIG. 6A), a hot water extract(FIG. 6B), a SCF CO₂ extract (FIG. 6C), and oleandrin (FIG. 6D).

FIG. 7 is a semilog plot of concentration of oleandrin versus percentgrowth of control of human melanoma BRO and PANC-1 cells forpolysaccharides isolated from the hot water extract.

DETAILED DESCRIPTION OF THE INVENTION

The extraction process can be conducted on a dried powder of Neriumoleander leaves prepared according to a process described in acurrently-pending U.S. provisional application Ser. No. 60/653,210 filedFeb. 15, 2005 in the name of Addington or U.S. application Ser. No.11/340,016 filed Jan. 26, 2006 in the name of Addington, the entiredisclosures of which are hereby incorporated by reference.

An important component of the method for processing oleander leaves isthe use of a patented comminuting and dehydrating system and methodwhich utilizes vortexes of air to extract moisture and separate theplant particles by size. Suitable comminuting and dehydrating systemsare described in U.S. Pat. No. 5,236,132, No. 5,598,979, No. 6,517,015,and No. 6,715,705, all to Frank Rowley, Jr., the entire disclosure ofeach of which is hereby incorporated by reference. In general, themethod for processing oleander leaves involved collecting suitableleaves and stems, washing the collected plant material, drying theleaves and stems, and passing the leaves through an apparatus which usesvortexes of air to extract moisture and separate the plant particles bysize. Larger particles were either re-processed or used as coarsematerial. The smallest particles were retained as fine oleander dustwhich can then be subjected to further extraction to obtain oleandrinand other pharmacologically active components.

Supercritical fluids are produced by heating a gas above its criticaltemperature or compressing a gas above its critical pressure.Supercritical fluid extraction comprises at least two steps: extractionand separation. An exemplary supercritical-fluid extractor comprises atank of the mobile phase, usually CO₂, a pump to pressurize the gas, anoven containing the extraction vessel, a restrictor to maintain a highpressure in the extraction line, and a trapping vessel. Analytes aretrapped by letting the solute-containing supercritical fluid decompressinto an empty vial, through a solvent, or onto a solid sorbent material.Extractions are done in dynamic, static, or combination modes. In adynamic extraction the supercritical fluid continuously flows throughthe sample in the extraction vessel and out the restrictor to thetrapping vessel. In static mode the supercritical fluid circulates in aloop containing the extraction vessel for some period of time beforebeing released through the restrictor to the trapping vessel. In thecombination mode, a static extraction is performed for some period oftime, followed by a dynamic extraction.

In general, the starting material is placed in an extractor devicetogether with the supercritical fluid at specified pressure andtemperature conditions to extract the desired components from the plantmaterial. After extraction, the fluid and the compound are passedthrough a separator which changes the pressure and temperature, therebyreducing the dissolving power of the supercritical fluid and causing theseparation or fractionation of the dissolved compound.

The SCF extract is prepared by mixing oleander plant starting materialwith carbon dioxide at a supercritical pressure and temperature, with orwithout a chemical modifier, then decreasing the pressure andtemperature of the mixture and separating out the extract. The extractis separated as the pressure and temperature of the mixture aredecreased. The use of powdered oleander leaves as a starting material ispreferred. The powdered leaf particles ensure that a maximum amount ofsurface and internal leaf area is exposed to the extraction process.This provides an exponential increase in the amount of active componentsthat are recovered in the extract, compared to methods of extractioncurrently available. The table below includes different solvents thatcan be used as the SCF extraction solvent and their correspondingcritical temperature and critical pressure.

Critical Critical Fluid Temperature (K) Pressure (bar) Carbon dioxide304.1 73.8 Ethane 305.4 48.8 Ethylene 282.4 50.4 Propane 369.8 42.5Propylene 364.9 46.0 Trifluoromethane (Fluoroform) 299.3 48.6Chlorotrifluoromethane 302.0 38.7 Trichlorofluoromethane 471.2 44.1Ammonia 405.5 113.5 Water 647.3 221.2 Cyclohexane 553.5 40.7 n-Pentane469.7 33.7 Toluene 591.8 41.0

Carbon dioxide is a preferred supercritical fluid for the extraction ofactive components from the oleander plant. Its critical temperature is31.06° C., its critical pressure is 73.83 bar, and its critical densityis 0.460 g/cm³. It is contemplated, however, that other compounds, ormixtures thereof, can be used in a SCF extraction process for oleandrin.

In some embodiments, a co-solvent or modifier is included in thesupercritical fluid. Modifiers generally possess volatility between thatof the supercritical fluid and of the compound being extracted, and theymust be miscible with the supercritical fluid. In some embodiments, themodifier is a liquid at ambient conditions. By way of example andwithout limitation, a modifier can be selected from the group consistingof ethanol, methanol, propanol, water, acetone, ethyl acetate, methylenechloride, etc. (See table above). For the extraction ofpharmacologically active components from the oleander plant, ethanol isa particularly suitable modifier. It can be used in a ratio of 35 to 75kg ethanol solvent per kg of biomass although the preferred ratio is 55kg solvent per kg biomass material. An exemplary extraction process forthe SCF extraction of oleandrin from Nerium oleander can be conducted asfollows or as detailed in Example 1. The starting comminuted plantmaterial is combined with the carbon dioxide in an extractor device.Pure CO₂, or a mixture thereof with one or more modifiers, is employedas the supercritical solvent. The extraction is conducted at a pressureof about 280 bar or about 270 to 320 bar, and a temperature of about 50°C. or about 40 to 60° C. The ratio of solvent to raw starting materialis preferably about 50:1 or about 45:1 to 60:1 based on weight of boththe solvent and the raw material.

In another exemplary extraction process, supercritical carbon dioxidefurther comprising ethanol as a modifier is added to the starting plantmaterial in an extractor device (see Example 1). The extraction isconducted at a pressure of about 280 bar (or about 270 to 320 bar), anda temperature of about 50° C. or about 40 to 60° C. The ratio of solventand modifier to raw starting material is preferably from about 40 toabout 45 to 1, based on the weight of both the solvent and modifiercombined and the raw material. The ethanol modifier is subsequentlyevaporated by use of vacuum.

Following extraction, separation is conducted. In some exemplaryembodiments, the supercritical solvent, with or without a modifier, incombination with the dissolved starting material, is passed through aseparator device which decreases the pressure and temperature of thesolvent mixture until the extract containing the active components isseparated and recovered.

The extract is a mixture of pharmacologically active compounds, such asoleandrin or other cardiac glycosides, and other plant material. Theoleandrin-rich extract obtained by the SCF process is a substantiallywater-insoluble, viscous semi-solid at ambient temperature. The SCFextract comprises many different components possessing a variety ofdifferent ranges of water solubility. Oleandrin extract from asupercritical fluid process contains by weight a theoretical range of0.9% to 2.5% oleandrin. SCF extracts comprising varying amount ofoleandrin have been obtained. In one embodiment, the SCF extractcomprises about 2% by wt. of oleandrin. The remainder of the viscoussemi-solid extract consists of water insoluble cellulose materials. Thehot-water extract has different properties than and a differentcomposition than the SCF extract. The SCF extract contains a 3-10 foldhigher concentration of oleandrin than the hot-water extract. This wasconfirmed by both HPLC as well as LC/MS/MS (tandem mass spectrometry)analyses.

The hot-water extract of the prior art was compared to the SCF extractof the invention. FIG. 1A depicts an HPLC chromatogram for each theprior art hot water extract, and FIG. 1B depicts an HPLC chromatogramfor each the SCF extract of the invention. The analysis was conducted asdetailed in Example 11. The peak at 17.6 min is identified as oleasideA. The peak at 29.9 min is identified as oleandrin. The chromatogramswere obtained by injecting samples of the extract at a concentration of10 mg extract/ml of HPLC buffer. The data for the hot-water extract wasobtained using a 30 μl injection volume and the data for the SCF extractwas obtained using a 10 μl injection volume. The samples assayed asfollows:

Extract Oleaside (%) Oleandrin (%) Hot-water 0.094 0.17 SCF 0.73 2.68

The two extract differ substantially in their concentration ofoleandrin, oleaside A and in the composition and relative amounts oftheir various other components that have not been identified herein. Theconcentration of oleandrin was increased by 15-fold due to thesupercritical CO₂ extraction process. As a potential clinical treatmentbenefit, much smaller amount of supercritical CO₂ extract will be neededto achieve similar activity and efficacy compared to the hot waterextract. As a result, the supercritical CO₂ extract is expected toprovide maximal therapeutic efficacy and overcome the over-dosagedrawback of hot water extract in order to achieve a similar therapeuticeffect.

The extracts also differ in their polysaccharide and carbohydratecontent. FIGS. 6A-6D depict HPLC chromatograms (with tandem MSdetection) for a carbohydrate reference sample (mixture of simplesaccharide and oligosaccharide standards ranging from a very simplesugar, d-xylose (molecular weight 150) to a relatively complexcarbohydrate, maltoheptaose (molecular weight 1152)), a hot waterextract, a SCF CO₂ extract, and oleandrin, respectively. The sampleswere analyzed according to Example 14. The reference sample includes amixture of monosaccarides and disaccharides. The hot water extractcontains 407.3 glucose equivalent units of carbohydrate relative to astandard curve prepared with glucose while analysis of the SCF CO₂extract found carbohydrate levels that were found in very low levelsthat were below the limit of quantitation. The amount of carbohydrate inthe hot water extract of Nerium oleander was, however, at least 100-foldgreater than that in the SCF CO₂ extract. The polysaccharide content ofthe SCF extract can be <0.5%, <0.1%, <0.05%, or <0.01% wt.

Carbohydrate content (μg glucose equivalents/mg of plant Nerium oleanderpreparation extract) Hot water extract 407.3 ± 6.3 SCF CO₂ extract BLQ(below limit of quantitation)

Based upon the findings above and LC/MS/MS data shown in FIG. 1, the SCFCO₂ extract contains no quantifiable sugar content in comparison to thehot water extract which is more than 40% carbohydrate by weight. It isextremely surprising that the SCF CO₂ extract, which contains noquantifiable amounts of polysaccharides in contrast to the hot waterextract, actually exhibits by 15 to 20-fold more antiproliferativeactivity than the hot water extract.

Contrary to the teachings of Ozel (supra), the antiproliferativeactivity of the hot water extract is not attributable to polysaccharidestherein. The polysaccharide components of the hot water extract wereisolated by subjecting the hot water extract to passage through a smallpreparative C18 solid phase column. This type of column only removesnon-water soluble (i.e. lipid soluble) components. As such, all watersoluble carbohydrates whether as simple sugars or as complexpolysaccharides simply pass through the column and are collected. Theaqueous, solution containing the collected carbohydrates, was thenbrought to dryness under nitrogen. The dried carbohydrate material fromthe hot water extract of Nerium oleander was then reconstituted in wateror tissue culture media and added to human melanoma cells to determinerelative cytotoxic potential using an MTT assay that measuresmitochondrial enzyme activity in live cells. This colorimetricmicrotiter based test therefore can be used to measure the relativeability of materials to inhibit proliferation of tumor cells. Therelative antiproliferative activity of the polysaccharide components ofthe hot water extract (isolated by Ozel et al), the intact hot waterextract and the total components of the instant SCF CO₂ extract werecompared using this method.

The data (see table below and FIG. 7) demonstrate that the components ofthe SCF CO₂ extract are extremely potent in inhibiting proliferation ofhuman tumor cell lines and therefore should be efficacious in treatingdiseases associated with excessive cell proliferation; whereas, thepolysaccharide components are not. In particular, once the cardiacglycosides (lipid soluble materials) are removed from the hot waterextract of Nerium oleander, the extract is without any significantpotential to inhibit the growth of malignant cells.

IC₅₀ (ug/ml)* Human BRO IC₅₀ (ug/ml) Nerium oleander product melanomaHuman Panc-1 pancreatic or component cancer cells cancer cellsSupercritical CO₂ extract 0.35 0.22 (SCF CO₂) Hot water extract 6.1 3.3**Hot water extract >100 >100 without lipid (cardiac glycoside)components *The IC₅₀ value is that concentration of product which whenincubated with cells in culture for a defined period of time (e.g. 72hr) produces 50% inhibition of cell proliferation or survival. **Theremoval of cardiac glycosides from the hot water extract leaves aproduct consisting almost entirely of simple as well as complex, that islinked, sugar resides. The later are also referred to as complexpolysaccharides.

The partial compositions of the SCF CO₂ extract and hot water extractwere determined by DART TOF-MS (Direct Analysis in Real Time Time ofFlight Mass Spectrometry) on a JEOL AccuTOF-DART mass spectrometer (JEOLUSA, Peabody, Mass., USA). The SCF extract was found to comprise some orall of the compounds selected from the group consisting of: oleandrinand oleandrigenin. The extract also comprised aglycone of odoroside andtriterpenes (such as described in Phytochem. 1995, 39, 171-174, theentire disclosure of which is hereby incorporated by reference). Othercompounds that might be present in the SCF extract include one or moreof neritaloside; oleanolic acid; ursolic acid; betulinic acid; betulin(urs-12-ene-3β,28-diol); 28-norurs-12-en-3β-ol; urs-12-en-3β-ol;3β,3β-hydroxy-12-oleanen-28-oic acid; 3β,20α-dihydroxyurs-21-en-38-oicacid; 3β,27-dihydroxy-12-ursen-38-oic acid;3β,13β-dihydroxyurs-11-en-28-oic acid;3β,12α-dihydroxyoleanan-28,13β-olide; 3β,27-dihydroxy-12-oleanan-28-oicacid; and other components. The hot water extract comprised high amountsof polysaccharide with only small amounts of oleandrin andoleandrigenin.

In some embodiments, the extract comprises one or more of the compoundslisted in the table below or otherwise herein. The following tableprovides a brief summary of suitable plant material sources that areextractable with SCF and the cardiac glycosides derived therefrom,wherein the cardiac glycosides have demonstrated efficacy against therespectively listed cell lines. Accordingly, a SCF extract of theinvention, or a pharmaceutical composition or dosage form comprising aSCF extract of the invention can also be used to treat the proliferativedisorders included in the table below.

Demstrated efficacy Plant Species Cardiac Glycoside(s) against ApocynumApocannoside, cymarin Human nasopharynx cannabinum L. carcinoma (KB)(Apocynaceae) Asclepias Calotropin, 16α-acetoxycalotropin, Human lungcarcinoma curassavica L. 15β-hydroxycalotropin, calactin, 15β- (A549),breast carcinomas (Asclepiadaceae) hydroxycalactin, asclepin, 16α-(MCF-7 and MDA-MB- hydroxyasclepin, uscharidin, uscharin, 231), andhepatoma uzarigenin (HepG2) Beaumontia Digitoxigenin, oleandrigenin,Human breast carcinoma brevituba Oliver digitoxigenin, α-L-cymaroside,(BC1), colon carcinoma (Apocynaceae) digitoxigenin β-gentiobiosyl-α-L-(Col2), fibrosarcoma (HT- cymaroside, Δ¹⁶-digitoxigenin β-D- 1080),nasopharyngeal glucosyl-α-L-cymaroside carcinoma (KB),vinblastine-resistant KB (KB-V1), lung carcinoma (Lu1), and melanoma(Mel2) Calotropis procera Calotropin, calactin, uscharin, Humannon-small-cell lung (Ait.) R. Br. voruscharin, 2″-oxovoruscharincarcinoma (A549), human (Asclepiadaceae) glioblastomas (Hs683 and U373),human colon carcinomas (HCT-15 and LoVo), hepatoma (Huh7), non-hepatoma(COS-1), and colorectal carcinoma (COLO 320) Cerbera odollam 2′-O-Acetylcerleaside A, 17α- Human oral epidermoid Gaertner neriifolin,17β-neriifolin, cerberin carcinoma (KB), breast (Apocynaceae) carcinoma(BC), and small- cell lung carcinoma (NCI- H187) Coronilla varia L.Hyrcanoside Human lymphocytic (Fabaceae) leukemia cells (P-388) andnasopharynx carcinomas (9KB) CrossopetalumSecurigenin-3β-O-β-6-deoxyguloside, Human oral epidermoid gaumeri(Loes.) 19-hydroxy-sarmentogenin-3β-O-β-6- carcinoma (KB) Lundelldeoxyguloside, sarmentogenin-3β-O- (Celastraceae)[α-allosyl-(1→4)-β-6-deoxyalloside],securigenin-3β-O-[α-allosyl-(1→4)-β- 6-deoxyalloside] Digitalis purpureaL. Digoxin, digitoxin, gitoxin Human prostate carcinomas(Scrophulariaceae) (LNCaP, DU145, PC3), Digitalis lanata renaladenocarcinoma (TK- (Scrophulariaceae) 10), breast adenocarcinoma(MCF-7), malignant melanoma (UACC-62), and chronic myelogenous leukemia(K-562) Elaeodendron sp. Elaeodendrosides Human ovarian carcinoma(A2780) Euonymus alata Acovenosigenin A 3-O-α-L- Human oral epidermoid(Thunb.) Sieb. ramnopyranoside, euonymoside A, (KB), promyelocytic(Celastraceae) euonymusoside A lymphoma (HL-60), non- small-cell lungcarcinoma (A549), and cervix carcinoma (Hela) Euonymus Euonymoside AHuman lung carcinoma sieboldianus (A549) and ovarian Blumeadenocarcinoma (SK-OV- (Celastraceae) 3) Maquira Maquiroside A Humanoral epidermoid calophylla (P.&E.) carcinoma (KB) C.C. Berg (Moraceae)Nerium oleander Oleander, oleandrin, cardenolide N-1, Human Jurkatleukaemia L. (Apocynaceae) cardenolide N-4,3β-O-(β-D- (T-cell),histiocytic sarmentosyl)-16β-acetoxy-14- lymphoma (U-937),hydroxy-5β,14β-card-20-(22)-enolide, promyelocytic lymphoma16β-acetoxy-3β,14-dihydroxy-5β,14β- (HL-60), cervicalcard-20-(22)-enolide carcinoma (Hela), breast carcinoma (MCF-7),prostate carcinomas (LNCap, DU145, PC3), malignant fibroblast (VA- 13),and liver carcinoma (HepG2) Nierembergia 17-epi-11α-hydroxy-6,7- Humanbreast carcinoma aristata D. Don dehydrostrophanthidin-3-O-β- (BC1),fibrosarcoma (HT), (Solanaceae) boivinopyranoside; 6,7- lung cancer(LU1), dehydrostrophanthidin-3-O-β- melanoma (Mel2), colonboivinopyranoside; 6,7- carcinoma (Col2), oraldehydrostrophanthidin-3-O-β- epidermoid (KB), drug oleandropyranosideresistant KB with and without vinblastine, epidermoid carcinoma (A-431), prostate carcinoma (LNCaP), hormone- dependent breast carcinoma(ZR-75-1), and glioma (U373) Ornithogalum Convallatoxin Human oralepidermoid umbellatum L. carcinoma (KB) (Hyacinthaceae) Pergularia3′-O-β-D-glucopyranosylcalactin, 12- Kaposis' sarcoma (KS) tomentosa L.dehydroxyghalakinoside, 6′- (Asclepiadaceae) dehydroxyghalakinoside,ghalakinoside, calactin Periploca graeca L. Periplocin isomers Humanprostate carcinoma (Asclepiadaceae) (PC-3) Rhodea japonica Rhodexin AHuman leukemia (K562) (Thunb.) Roth. (Liliaceae) Saussurea stella3-O-β-D-fucopyranosylstrophanthidin, Human gastric cancer Maxim.3-O-β-D- (BGC-823) and hepatoma (Asteraceae)quinovopyranosylperiplogenin, 3-O-β- (Bel-7402)D-glucopyranosyl-(1→4)-α-L- rhamnopyranosylcannogenin, 3-O-β-D-xylopyranosylperiplogenin, 3-O-β- D-quinovopyranosylstrophanthidin, 3-O-β-D-xylopyranosylstrophanthidin, 3-O-β-D-fucopyranosylperiplogenin,3-O-α-L-rhamnopyranosylcannogenol, convallatoxin, 3-O-α-L-rhamnpyranosylacovenosigenin A Streblus asper Stebloside, mansonin Oralhuman epidermoid Lour. (Moraceae) carcinoma (KB) StreptocaulonPeriplogenin digitoxoside, Human fibrosarcoma (HT- juventas (Lour.)Periplocymarin, digitoxigenin 3-O-[O- 1080) Merr.β-D-glucopyranosyl-(1→6)-O-β-D- (Asclepiadaceae)glucopyranosyl-(1→4)-β-D- digitoxopyranoside, echujin, corchorusoside CStreptocaulon 3-O-(β- Human gastrointestinal griffithii Hook.f.glucopyranosyl)acovenosigenin A cancer (HCG-27), lung (Asclepiadaceae)carcinoma (A549), breast carcinoma (MCF-7), and cervical carcinoma(HeLa) Strophanthus Ouabain Human prostate carcinomas (LNCaP, DU145,PC3) Thevetia ahouia Neriifolin, 3′-O-methylevomonoside, National CancerInstitute's (L.) A. DC. 2′acetylneriifolin human disease oriented 60-(Apocynaceae) cell line tumor screening panel Thevetia peruvianaThevetin A and B, thevetoside Human hepatoma (SMMC- (Pers.) K. Schum.7721), gastric carcinoma (Apocynaceae) (SGC-7901), and cervicalcarcinoma (HeLa) Urginea maritime Proscillaridin A, scillaren A Humanbreast carcinoma (L.) Baker (MCF-7) (Liliaceae)

A cardiac glycoside obtained by SCF extraction can be subsequentlymodified to form an extract or composition comprising a semisyntheticcardiac glycoside. For example, a cardiac glycoside (oxovoruscharin,UNBS-1244) originally isolated from an African plant Calotropis proceracan be modified to form semisynthetic UNBS-1450 (Mijatovic et al. DrugDev. Res. (2007), 68(4), 164-173, and Mol. Cancer. Ther. (2006), 5(2),391-399, the entire disclosures of which are hereby incorporated byreference).

The extracts also differ in their relative performance as determined byefficacy against several tumor cell lines (Example 10).Oleandrin-containing samples were prepared to contain the same amount ofoleandrin although the concentration of oleandrin in each sample varieddue to the differences in the concentration of in the extracts. The dataobtained are summarized in the table below and in FIGS. 4 and 5.

Human melanoma Human pancreatic cancer DRUG BRO cells (IC₅₀, μM) PANC-1cells (IC₅₀, μM) Oleandrin 0.017* 0.01 Hot water extract 0.052 0.03Supercritical 0.007 0.004 CO₂ extract *The IC₅₀ of tested compounds arepresented as micromolar (μM) oleandrin concentration in those extracts.That is, the data represent that concentration of oleandrin as freechemical or as part of an extract necessary to inhibit growth orproliferation of tumor cell growth compared to untreated cells by 50%.

As shown in the table above, the IC₅₀ value of the supercritical CO₂extract is only 50% of that oleandrin alone in both Panc-1 and BROcells, which suggested that the supercritical CO₂ extract of oleander isat least two-fold stronger (more potent) than oleandrin alone withrespect to the inhibition of the growth of Panc-1 or BRO cells. Incomparison, hot water extract was the least potent among three entitiestested. The data demonstrate potent cytotoxicity against human tumorcell lines by oleandrin as well as the extracts with the relativepotency occurring as follows: supercritical CO₂ extract> oleandrin> hotwater extract. These data imply that the cytotoxicity of thesupercritical CO₂ extract is probably due to the presence of at leastone other pharmacologically active component in the SCF extract inaddition to oleandrin and that the potency of the supercritical CO₂extract is much greater (7.4 fold) than that of the hot water extract.The data (FIGS. 4 and 5) clearly demonstrate the substantial improvementin efficacy of the SCF extract over the hot-water extract and evenoleandrin alone. The improvement in efficacy exceeded the expectedimprovement that might have been obtained based solely upon theincreased concentration of oleandrin in the SCF extract.

Phosphorylation of the serine/threonine kinase known as Akt providestumor cells with enhanced survival capability. Increased Akt activitypromotes survival of tumor cells that would normally undergo death byapoptosis. In addition, pAkt is involved in cell proliferation,angiogenesis, genome instability and cell invasion and migration(Yoeli-Lerner M and Toker A. Akt/PKB Signaling in Cancer. Cell Cycle5:603-605, 2006). All of these responses contribute to initiation andprogression. Further evidence of the importance of Akt signaling incancer comes from studies which have detected over-expression andhyper-activation (through phosphorylation) of Akt in a wide range ofhuman tumors, and this is often linked with poor prognosis. The relativeactivity of the hot-water and SCF extract on critical cell signalingproteins in human pancreatic cancer (Panc-1) cells were also compared.The data (FIG. 3) demonstrate a decreased activation(concentration-dependent decline in expression of the phosphorylatedform pAkt) of protein kinase Akt and an increased activation of theMAPK/ERK (mitogen-activated protein kinase/extracellularsignal-regulated kinase) pathway (concentration dependent increase inphosphorylated form pERK). Both oleandrin and supercritical CO₂ extractwere capable of inhibiting PI3 Kinase resulting in reduction ofphosphorylation of Akt in Panc-1 cells, whereas the hot water extractdid not show this activity. Additionally, the expression of pERK wasdramatically increased in cells treated with either oleandrin orsupercritical CO₂ extract, but not in the cells treated with hot waterextract. The relative ability of the supercritical CO₂ extract toinhibit pAkt expression is much greater than that of oleandrin or thehot water extract of Nerium oleander. Given the fact that phospho-Akthas been associated with cancer cell survival and increased drug andradiation resistance to cancer cells, inhibition of pAkt would lead toinhibition of proliferation of cancer cells. Therefore, these resultssuggest that supercritical CO₂ extract has a very similar mechanism ofinhibition of proliferation of Panc-1 cells by suppressing theexpression of pAkt and increasing the expression of pERK, but the effectis much stronger than that of oleandrin alone. We did not observe anysimilar changes in the cells treated with hot water extract.Accordingly, the invention provides a method of inhibiting or reducingthe extent of Akt phosphorylation in a cancer cell by treating the cellwith an effective amount of SCF extract of the invention. In someembodiments, the effective amount of extract is that amount equivalentto that containing an equivalent of at least 5 nM although a range of 5to 50 nM is considered useful. Such a concentration of supercriticalextract will be useful in terms of inhibiting tumor cell proliferationas well as tumor cell migration and metastases. In addition, inhibitionof pAkt will prevent angiogenesis and, thus, tumor cell proliferationthrough inhibition of the development of blood and nutrient supply tothe growing tumor.

The invention also provides a method of enhancing the expression of pERK(extracellular-signal-regulated kinase; ERK) in a cancer cell bytreating the cell with an effective amount of SCF extract of theinvention. In some embodiments, the effective amount of extract is atleast 5 nM but a range of 5 nM to 50 nM is considered useful. Activationof ERK through phosphorylation is required for induction of autophagictumor cell death and in addition leads to induction of p21, a proteininvolved in cell cycle arrest (inhibition of proliferation of tumorcells).

The invention also provides a method of inhibiting the proliferation ofcancer cells by treatment of the cells with an effective of amount SCFextract of the invention.

The effect of oleandrin, the hot-water and SCF extract upon cell cyclechanges of PANC-1 cells treated therewith was evaluated over a 24-hourperiod. Panc-1 cells were treated with 25 nM of oleandrin alone or theamount of hot water extract or supercritical CO₂ extract of oleanderwhich was equivalent to 25 nM of oleandrin for 24 hrs. Cell cycleanalysis was carried out by flow cytometry. Cell division consists oftwo consecutive processes, mainly characterized by DNA replication andthen segregation of replicated chromosomes into two separate cells.Replication of DNA occurs in a specific part of the interphase called Sphase. S phase is preceded by a gap called G1 during which the cell ispreparing for DNA synthesis. This is 5 then followed by a gap called G2during which the cell prepares for mitosis. Then this is followed withthe mitosis phase, or M phase. G1, S, G2 and M phases are thetraditional subdivisions of the standard cell cycle. Cells which are ina G2/M block such as that induced by the supercritical CO2 extractcannot undergo division. The data are summarized in the table below.

Compounds G1 phase S phase G2/M phase Control 37.0 47.7 15.4 Oleandrin30.5 43.3 26.2 Hot water 33.0 38.8 23.2 extract Supercritical 30.1 40.229.7 CO₂ extractThe data are expressed as the relative percentage of cells in a givenphase of the cell cycle.

The data demonstrate that oleandrin as well as the other two oleanderextracts all inhibit the proliferation of panc-1 cells through causingcells to arrest at the G₂/M phase. Again, the supercritical CO₂ extractat a similar concentration led to a stronger G₂/M phase arrest comparedto oleandrin alone or hot water extract.

Based upon the data herein, the present inventors have demonstrated thatthe supercritical CO₂ extract can be specifically formulated to achievea useful level of oral bioavailability. No such data is available fororal absorption of the prior art hot water extract.

As evidenced by the data herein, the SCF extract comprises a mixture ofvarious components. Some of those components include oleandrin, oleasideA, oleandrigenin, neritaloside and odorside (Wang X, Plomley J B, NewmanR A and Cisneros A. LC/MS/MS analyses of an oleander extract for cancertreatment. Alanytical Chem. 72: 3547-3552, 2000) and other unidentifiedcomponents. The SCF extractable unidentified components of the SCFextract appear to include at least one other pharmacologically activecomponent that contributes to the efficacy of the oleandrin in the SCFextract. The at least one other SCF extractable component functionsadditively or synergistically with the oleandrin to provide the observedefficacy.

Patients undergoing a therapeutic regimen with the hot water extract arerequired to self-administer a daily intramuscular bolus. Practitionersof the instant invention in a clinical setting could expect increasedpatient compliance with a treatment regimen when compared to that of anintramuscular route of administration. The practitioners could alsoexpect increased acceptability (in terms of compliance) for the oralroute of administration to subjects for long term therapy when comparedto the daily intramuscular route of administration by intramuscularinjection. The practitioners could also expect an improved ability todose titrate the SCF extract as compared to a hot water extract sincethe hot water extract has limitations determined by the volume of thebolus. To the knowledge of the present inventors, no such limitationsexist in the instant invention.

The formulation and pharmaceutical composition of the inventioncomprises an SCF extract of Nerium species and an extract-solubilizingamount of solubilizer. As used herein, the term “solubilizer” means acompound, or mixture of compounds, that aids in the dissolution,emulsification, or dispersion of one or more components, at leastoleandrin, of the SCF extract in an aqueous environment. A solubilizercomprises one, two, three or more materials selected from the groupconsisting of a water soluble (miscible) co-solvent, a water insoluble(immiscible) co-solvent, an antioxidant, liquid carrier, surfactant anda combination thereof. Exemplary solubilizers include, by way of exampleand without limitation, those compounds disclosed in U.S. Pat. No.6,451,339, the entire disclosure of which is hereby incorporated byreference. As used herein, the term “extract-solubilizing amount” refersto an amount of solubilizer sufficient to dissolve at least asubstantial portion (at least 5% wt. or at least 25% wt. or at least 50%wt.) of the extract when the pharmaceutical composition is placed in anaqueous medium for a sufficient period of time, e.g. at least 10, atleast 20 or at least 30 minutes. The solubilizer can comprise one, two,three, four, five or more excipients. The solubilizer can serve as a“solubilizing agent”, meaning a compound, or mixture of compounds, thataids in dissolution of one or more components, at least oleandrin oranother pharmacologically active agent, of the SCF extract in an aqueousenvironment. The solubilizer can also serve as an “emulsifying agent”,meaning a compound, or mixture of compounds, that aids in emulsificationof one or more components, at least oleandrin or anotherpharmacologically active agent, of the SCF extract in an aqueousenvironment.

It should be noted that a compound herein might possess one or morefunctions in the formulation of the invention. For example, a compoundmight serve as both a surfactant and a water miscible solvent or as botha surfactant and a water immiscible solvent.

Exemplary combinations of excipients in the solubilizer include at leastthe following: a) at least one water miscible solvent, at least oneantioxidant, and at least one surfactant; b) at least one water misciblesolvent and at least one surfactant; c) at least one water immisciblesolvent, at least one water miscible solvent, at least one antioxidant,and at least one surfactant; and d) other combinations of two, three,four, five or more excipients.

Depending upon the combination of materials in the solubilizer, theliquid pharmaceutical composition can form a solution, micelle emulsion,dispersion, microparticulate or solid dispersion when placed in anaqueous environment, such as an assay solution or the GI tract of asubject following oral administration.

The liquid composition can comprise one or more pharmaceuticallyacceptable liquid carriers. The liquid carrier can be an aqueous,non-aqueous, polar, non-polar, and/or organic carrier. Liquid carriersinclude, by way of example and without limitation, a water misciblesolvent, water immiscible solvent, water, buffer and mixtures thereof.

As used herein, the terms “water soluble solvent” or “water misciblesolvent”, which terms are used interchangeably, refer to an organicliquid which does not form a biphasic mixture with water or issufficiently soluble in water to provide an aqueous solvent mixturecontaining at least five percent of solvent without separation of liquidphases. The solvent is suitable for administration to humans or animals.Exemplary water soluble solvents include, by way of example and withoutlimitation, PEG (poly(ethylene glycol)), PEG 400 (poly(ethylene glycolhaving an approximate molecular weight of about 400), ethanol, acetone,alkanol, alcohol, ether, propylene glycol, glycerin, triacetin,poly(propylene glycol), PVP (poly(vinyl pyrrolidone)),dimethylsulfoxide, N,N-dimethylformamide, formamide,N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol,soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

As used herein, the terms “water insoluble solvent” or “water immisciblesolvent”, which terms are used interchangeably, refer to an organicliquid which forms a biphasic mixture with water or provides a phaseseparation when the concentration of solvent in water exceeds fivepercent. The solvent is suitable for administration to humans oranimals. Exemplary water insoluble solvents include, by way of exampleand without limitation, medium/long chain triglycerides, oil, castoroil, corn oil, vitamin E, vitamin E derivative, oleic acid, fatty acid,olive oil, softisan 645 (Diglyceryl Caprylate/Caprate/Stearate/Hydroxystearate adipate), miglyol, captex (Captex 350: GlycerylTricaprylate/Caprate/Laurate triglyceride; Captex 355: GlycerylTricaprylate/Caprate triglyceride; Captex 355 EP/NF: GlycerylTricaprylate/Caprate medium chain triglyceride).

Suitable solvents are listed in the “International Conference onHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH) guidance for industry Q3CImpurities: Residual Solvents” (1997), which makes recommendations as towhat amounts of residual solvents are considered safe inpharmaceuticals. Preferred solvents are listed as class 2 or class 3solvents. Class 3 solvents include, for example, acetic acid, acetone,anisole, 1-butanol, 2-butanol, butyl acetate, tert-butlymethyl ether,cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid,heptane, isobutyl acetate, isopropyl acetate, methyl acetate,methyl-1-butanol, methylethyl ketone, methylisobutyl ketone,2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, orpropyl acetate.

Other materials that can be used as water immiscible solvents in theinvention include: Captex 100: Propylene Glycol Dicaprate; Captex 200:Propylene Glycol Dicaprylate/Dicaprate; Captex 200 P: Propylene GlycolDicaprylate/Dicaprate; Propylene Glycol Dicaprylocaprate; Captex 300:Glyceryl Tricaprylate/Caprate; Captex 300 EP/NF: GlycerylTricaprylate/Caprate Medium Chain Triglycerides; Captex 350: GlycerylTricaprylate/Caprate/Laurate; Captex 355: Glyceryl Tricaprylate/Caprate;Captex 355 EP/NF: Glyceryl Tricaprylate/Caprate Medium ChainTriglycerides; Captex 500: Triacetin; Captex 500 P: Triacetin(Pharmaceutical Grade); Captex 800: Propylene Glycol Di(2-Ethylhexanoate); Captex 810 D: GlycerylTricaprylate/Caprate/Linoleate; Captex 1000: Glyceryl Tricaprate; CaptexCA: Medium Chain Triglycerides; Captex MCT-170: Medium ChainTriglycerides; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP/NF:Glyceryl Monooleate; Capmul MCM: Medium Chain Mono- & Diglycerides;Capmul MCM C8: Glyceryl Monocaprylate; Capmul MCM C10: GlycerylMonocaprate; Capmul PG-8: Propylene Glycol Monocaprylate; Capmul PG-12:Propylene Glycol Monolaurate; Caprol 10G10O: Decaglycerol Decaoleate;Caprol 3GO: Triglycerol Monooleate; Caprol ET: Polygycerol Ester ofMixed Fatty Acids; Caprol MPGO: Hexaglycerol Dioleate; Caprol PGE 860:Decaglycerol Mono-, Dioleate.

As used herein, a “surfactant” refers to a compound that comprises polaror charged hydrophilic moieties as well as non-polar hydrophobic(lipophilic) moieties; i.e., a surfactant is amphiphilic. The termsurfactant may refer to one or a mixture of compounds. A surfactant canbe a solubilizing agent, an emulsifying agent or a dispersing agent.

An empirical parameter commonly used to characterize the relativehydrophilicity and hydrophobicity of non-ionic amphiphilic compounds isthe hydrophilic-lipophilic balance (“HLB” value). Surfactants with lowerHLB values are more hydrophobic, and have greater solubility in oils,while surfactants with higher HLB values are more hydrophilic, and havegreater solubility in aqueous solutions. Using HLB values as a roughguide, hydrophilic surfactants are generally considered to be thosecompounds having an HLB value greater than about 10, as well as anionic,cationic, or zwitterionic compounds for which the HLB scale is notgenerally applicable. Similarly, hydrophobic surfactants are compoundshaving an HLB value less than about 10.

It should be appreciated that the HLB value of a surfactant is merely arough guide generally used to enable formulation of industrial,pharmaceutical and cosmetic emulsions. For many important surfactants,including several polyethoxylated surfactants, it has been reported thatHLB values can differ by as much as about 8 HLB units, depending uponthe empirical method chosen to determine the HLB value (Schott, J.Pharm. Sciences, 79(1), 87-88 (1990)). Likewise, for certainpolypropylene oxide containing block copolymers (PLURONIC surfactants,BASF Corp.), the HLB values may not accurately reflect the true physicalchemical nature of the compounds. Finally, commercial surfactantproducts are generally not pure compounds, but are complex mixtures ofcompounds, and the HLB value reported for a particular compound may moreaccurately be characteristic of the commercial product of which thecompound is a major component. Different commercial products having thesame primary surfactant component can, and typically do, have differentHLB values. In addition, a certain amount of lot-to-lot variability isexpected even for a single commercial surfactant product. Keeping theseinherent difficulties in mind, and using HLB values as a guide, oneskilled in the art can readily identify surfactants having suitablehydrophilicity or hydrophobicity for use in the present invention, asdescribed herein.

The hydrophilic surfactant can be any hydrophilic surfactant suitablefor use in pharmaceutical compositions. Such surfactants can be anionic,cationic, zwitterionic or non-ionic, although non-ionic hydrophilicsurfactants are presently preferred. As discussed above, these non-ionichydrophilic surfactants will generally have HLB values greater thanabout 10. Mixtures of hydrophilic surfactants are also within the scopeof the invention.

Similarly, the hydrophobic surfactant can be any hydrophobic surfactantsuitable for use in pharmaceutical compositions. In general, suitablehydrophobic surfactants will have an HLB value less than about 10.Mixtures of hydrophobic surfactants are also within the scope of theinvention.

The choice of specific hydrophobic and hydrophilic surfactants should bemade keeping in mind the particular hydrophobic therapeutic agent to beused in the composition, and the range of polarity appropriate for thechosen therapeutic agent, as discussed in more detail below. With thesegeneral principles in mind, a very broad range of surfactants issuitable for use in the present invention. Such surfactants can begrouped into the following general chemical classes detailed in theTables below. The HLB values given in the Tables below generallyrepresent the HLB value as reported by the manufacturer of thecorresponding commercial product. Incases where more than one commercialproduct is listed, the HLB value is the Tables is the value as reportedfor one of the commercial products, a rough average of the reportedvalues, or a value that, in the judgment of the present inventors, ismore reliable. It should be emphasized that the invention is not limitedto the surfactants in the following Tables, which show representative,but not exclusive, lists of available surfactants.

1. Polyethoxylated Fatty Acids

Although polyethylene glycol (PEG) itself does not function as asurfactant, a variety of PEG-fatty acid esters have useful surfactantproperties. Among the PEG-fatty acid monoesters, esters of lauric acid,oleic acid, and stearic acid are most useful. Among the surfactants ofTable 1, preferred hydrophilic surfactants include PEG-8 laurate, PEG-8oleate, PEG-8 stearate, PEG-9 oleate, PEG-10 laurate, PEG-10 oleate,PEG-12 laurate, PEG-12 oleate, PEG-15 oleate, PEG-20 laurate and PEG-20oleate. Examples of polyethoxylated fatty acid monoester surfactantscommercially available are shown in Table 1.

TABLE 1 PEG-Fatty Acid Monoester Surfactants Compound Commercial Product(Supplier) HLB PEG 4-100 Crodet L series (Croda) >9 monolaurate PEG4-100 Crodet O series (Croda) >8 monooleate PEG 4-100 Crodet S series(Croda), Myrj Series >6 monostearate (Atlas/ICI) PEG 400 distearateCithrol 4DS series (Croda) >10 PEG 100, 200, 300 Cithrol ML series(Croda) >10 monoleaurate PEG 100, 200, 300 Cithrol MO series (Croda) >10monooleate PEG 400 dioleate Cithrol 4DO series (Croda) >10 PEG 400-100OCithrol MSseries (Croda) >10 monostearate PEG-1 stearate Nikkol MYS-1EX(Nikko), Coster K1 2 (Condea) PEG-2 stearate Nikkol MYS-2 (Nikko) 4PEG-2 oleate NIkkol MYO-2 (Nikko) 4.5 PEG-4 laurate Mapeg 200 ML (PPG),Kessco PEG 200 ML 9.3 (Stepan), LIPOPEG 2L (LIPO Chem.) PEG-4 oleateMapeg 200 MO (PPG), KEssco PEG200 MO 8.3 (Stepan) PEG-4 stearate KesscoPEG 200 MS (Stepan), Hodag 20 S 6.5 (Calgene), Nikkol MYS-4 (Nikko)PEG-5 stearate Nikkol TMGS-5 (Nikko) 9.5 PEG-5 oleate NIkkol TMGO-5(Nikko) 9.5 PEG-6 oleate Algon OL 60 (Auschem SpA), Kessco 8.5 PEG 300MO (Stepan), Nikkol MYO-6 (Nikko), Emulgante A6 (Condea) PEG-7 oleateAlgon OL 70 (Auschem SpA) 10.4 PEG-6 laurate Kessco PEG300 ML (Stepan)11.4 PEG-7 laurate Lauridac 7 (Condea) 13 PEG-6 sterate Kessco PEG300 MS(Stepan) 9.7 PEG-8 laurate Mapeg 400 ML (PPG), LIPOPEG 13 4DL (LipoChem.) PEG-8 oleate Mapeg 400 MO (PPG), Emulgante A8 12 (Condea) PEG-8stearate Mapeg 400 MS (PPG), Myrj 45 12 PEG-9 oleate Emulgante A9(Condea) >10 PEG-9 stearate Cremophor S9 (BASF) >10 PEG-10 laurateNikkol MYL-10 (Nikko), Lauridac 10 13 (Croda) PEG-10 oleate NIkkolMYO-10 (Nikko) 11 PEG-10 stearate NikkolMYS-10 (Nikko), Coster K100 11(Condea) PEG-12 laurate Kessco PEG 600 ML (Stepan) 15 PEG-12 oleateKessco PEG 600 MO (Stepan) 14 PEG-12 ricinoleate (CAS #9004-97-1) >10PEG-12 stearate Mapeg 600 MS (PPG), Kessco PEG 600 MS 14 (Stepan) PEG-15stearate MIkkol TMGS-15 (Nikko), Koster K15 14 (Condea) PEG-15 oleateNikkol TMGO-15 (Nikko) 15 PEG-20 laurate Kessco PEG 1000 ML (Stepan) 17PEG-20 oleate Kessco PEG 1000 MO (Stepan) 15 PEG-20 stearate Mapeg 1000MS (PPG), Kessco PEG 1000 16 MS (Stepan), Myrj 49 PEG-25 stearate NikkolMYS-25 (Nikko) 15 PEG-32 laurate Kessco PEG 1540 ML (Stepan) 16 PEG-32oleate Kessco PEG 1540 MO (Stepan) 17 PEG-32 stearate Kessco PEG 1540 MS(Stepan) 17 PEG-30 stearate Myrj 51 >10 PEG-40 laurate Crodet L40(Croda) 17.9 PEG-40 oleate Crodet O40 (Croda) 17.4 PEG-40 stearate Myrj52, Emerest 2715 (Henkel, Nikkol >10 MYS-40 (Nikko) PEG-45 stearateNIkkol MYS-45 (Nikko) 18 PEG-50 stearate Myrj 53 >10 PEG-55 stearateNikkol MYS-55 (Nikko) 18 PEG-100 oleate Crodet O-100 (Croda) 18.8PEG-100 stearate Myrj 59, Arlacel 165 (ICI) 19 PEG-200 oleate Albunol200 MO (Taiwan Surf.) >10 PEG-400 oleate LACTOMUL (Henkel), Albunol 400MO >10 (Taiwan Surf.) PEG-600 oleate Albunol 600 MO (Taiwan Surf.) >10

2. PEG-Fatty Acid Diesters

Polyethylene glycol fatty acid diesters are also suitable for use assurfactants in the compositions of the present invention. Among thesurfactants in Table 2, preferred hydrophilic surfactants, includePEG-20 dilaurate, PEG-20 dioleate, PEG-20 distearate, PEG-32 dilaurateand PEG-32 dioleate. Representative PEG-fatty acid diesters are shown inTable 2.

TABLE 2 PEG-Fatty Acid Diester Surfactants Compound Commercial Product(Supplier) HLB PEG-4 dilaurate Mapeg 200 DL (PPG), Kessco PEG 7 200 DL(Stepan), LIPOPEG 2-DL (Lipo Chem.) PEG-4 dioleate Mapeg 200 DO (PPG) 6PEG-4 distearate Kessco 200 DS (Stepan) 5 PEG-6 dilaurate Kessco PEG 300DL (Stepan) 9.8 PEG-6 dioleate Kessco PEG 300 DO (Stepan) 7.2 PEG-6distearate Kessco PEG 300 DS (Stepan) 6.5 PEG-8 dilaurate Mapeg 400 DL(PPG), Kessco PEG 400 DL 11 (Stepan), LIPOPEG 4 DL (Lipo Chem.) PEG-8dioleate Mapeg 400 DO (PPG), Kessco PEG 400 DO 8.8 (Stepan), LIPOPEG 4DO (Lipo Chem.) PEG-8 distearate Mapeg 400 DS (PPG), CDS 400 (Nikkol) 11PEG-10 dipalmitate Polyaldo 2PKFG >10 PEG-12 dilaurate Kessco PEG 600 DL(Stepan) 11.7 PEG-12 distearate Kessco PEG 600 DS (Stepan) 10.7 PEG-12dioleate Mapeg 600 DO (PPG), Kessco 600 DO 10 (Stepan) PEG-20 dilaurateKessco PEG 1000 DL (Stepan) 15 PEG-20 dioleate Kessco PEG 1000 DO(Stepan) 13 PEG-20 distearate Kessco PEG 1000 DS (Stepan) 12 PEG-32dilaurate Kessco PEG 1540 DL (Stepan) 16 PEG-32 dioleate Kessco PEG 1540DO (Stepan) 15 PEG-32 distearate Kessco PEG 1540 DS (Stepan) 15 PEG-400dioleate Cithrol 4DO series (Croda) >10 PEG-400 disterate Cithrol 4DSseries (Croda) >10

3. PEG-Fatty Acid Mono- and Di-Ester Mixtures

In general, mixtures of surfactants are also useful in the presentinvention, including mixtures of two or more commercial surfactantproducts. Several PEG-fatty acid esters are marketed commercially asmixtures or mono- and diesters. Representative surfactant mixtures areshown in Table 3.

TABLE 3 PEG-Fatty Acid Mono- and Diester Mixtures Compound CommercialProduct (Supplier) HLB PEG 4-150 mono, Kessco PEG 200-6000 mono,dilaurate N/A dilaurate (Stepan) PEG 4-150 mono, Kessco PEG 200-6000mono, dioleate (Stepan) N/A dioleate PEG 4-150 mono, Kessco PEG 200-6000mono, distearate N/A Distearate (Stepan)

4. Polyethylene Glycol Glycerol Fatty Acid Esters

Suitable PEG glycerol fatty acid esters are shown in Table 4. Among thesurfactantsin the Table, preferred hydrophilic surfactants are PEG-20glyceryl laurate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate,PEG-20 glyceryl oleate, and PEG-30 glyceryl oleate.

TABLE 4 PEG Glycerol Fatty Acid Esters Compound Commercial Product(Supplier) HLB PEG-20 glyceryl laurate Tagat L (Goldschmidt) 16 PEG-30glyceryl laurate Tagat L2 (Goldschmidt) 16 PEG-15 glyceryl laurateGlycerox L series (Croda) 15 PEG-40 glyceryl stearate Glycerox L series(Croda) 15 PEG-20 glyceryl stearate Capmul EMG (ABITEC), 13 Aldo MS-20KFG (Lonza) PEG-20 glyceryl oleate Tagat O (Goldschmidt) >10 PEG-30glyceryl oleate Tagat O2 (Goldschmidt) >10

5. Alcohol Oil Transesterification Products

A large number of surfactants of different degrees of hydrophobicity orhydrophilicity can be prepared by reaction of alcohols or polyalcoholswith a variety of natural and/or hydrogenated oils. Most commonly, theoils used are castor oil or hydrogenated castor oil, or an ediblevegetable oil such as corn oil, olive oil, peanut oil, palm kernel oil,apricot kernel oil, or almond oil. Preferred alcohols include glycerol,sorbitol, and pentaerythritol. Among these alcohol-oil transesterifiedsurfactants, preferred hydrophilic surfactants are PEG-35 castor oil(Incrocas-35), PEG-40 hydrogenated castor oil (Cremophor RH 40), PEG-25trioleate (TAGAT TO), PEG-60 corn glycerides (Crovol M70), PEG-60 almondoil (Crovol A70), PEG-40 palm kernel oil (Crovol PK70), PEG-50 castoroil (Emalex C-50), PEG-50 hydrogenated castor oil (Emalex HC-50), PEG-8caprylic/capric glycerides (Labrasol), and PEG-6 caprylic/capricglycerides (Softigen 767). Preferred hydrophobic surfactants in thisclass include PEG-5 hydrogenated castor oil, PEG-7 hydrogenated castoroil, PEG-9 hydrogenated castor oil, PEG-corn oil (Labrifil M 2125 CS),Peg-6 almond oil (Labrifil M 1944 CS), PEG-6 olive oil (Labrifil M 1980CS), PEG-6 peanut oil (Labrifil M 1969 CS), PEG-6 hydrogenated palmkernel oil (Labrifil M 2130 BS), PEG-6 triolein (Labrifil b M 2735 CS),PEG-8 corn oil (Labrifil WL 2609 BS), PEG-20 corn glycerides (CrovelM40), and PEG-20 almond glycerides (Crovel A40). The latter twosurfactants are reported to have HLB values of 10, which is generallyconsidered to be the approximate border line between hydrophilic andhydrophobic surfactants. For purposes of the present invention, thesetwo surfactants are considered to by hydrophobic. Representativesurfactants of this class suitable for use in the present invention areshown in Table 5.

TABLE 5 Transesterification Products of Oils and Alcohols CompoundCommercial Product (Supplier) HLB PEG-3 castor oil Nikkol CO-3 (Nikko) 3PEG-5, 9, and 16 castor oil ACCONON CA series (ABITEC) 6-7 PEG-20 castoroil Emalex C-20 (Nihon Emulsion), Nikkol CO-20 11 PEG-23 castor oilEmulgante EL23 >10 PEG-30 castor oil Emalex C-30 (Nihon Emulsion),Alkamuls 11 EL 620 (Rhone-Poulenc), Incrocas 30 (Croda) PEG-35 castoroil Cremophor EL and EL-P (BASF), Emulphor EL, N/A Incrocas-35 (Croda),Emulgin RO 35 (Henkel) PEG-38 castor oil Emulgante EL 65 (Condea) PEG-40castor oil Emalex C-40 (Nihon Emulsion), Alkamuls EL 719 13(Rhone-Poulenc) PEG-50 castor oil Emalex C-50 (Nihon Emulsion) 14 PEG-56castor oil Eumulgin PRT 56 (Pulcra SA) >10 PEG-60 castor oil NikkolCO-60TX (Nikko) 14 PEG-100 castor oil Thornley >10 PEG-200 castor oilEumulgin PRT 200 (Pulcra SA) >10 PEG-5 hydrogenated Nikkol HCO-5 (Nikko)6 castor oil PEG-7 hydrogenated Simusol 989 (Seppic), Cremophor WO7(BASF) 6 castor oil PEG-10 hydrogenated Nikkol HCO-10 (Nikko) 6.5 castoroil PEG-20 hydrogenated Nikkol HCO-20 (Nikko) 11 castor oil PEG-25hydrogenated Simulsol 1292 (Seppic), Cerex ELS 250 11 castor oil(Auschem SpA) PEG-30 hydrogenated Nikkol HCO-30 (Nikko) 11 castor oilPEG-40 hydrogenated Cremophor RH 40 (BASF), Croduret (Croda), 13 castoroil Emulgin HRE 40 (Henkel) PEG-45 hydrogenated Cerex ELS 450 (AuschemSpa) 14 castor oil PEG-50 hydrogenated Emalex HC-50 (Nihon Emulsion) 14castor oil PEG-60 hydrogenated Nikkol HCO060 (Nikko); Cremophor 15castor oil RH 60 (BASF) PEG-80 hydrogenated Nikkol HCO-80 (Nikko) 15castor oil PEG-100 hydrogenated Nikkol HCO-100 (Nikko) 17 castor oilPEG-6 corn oil Labrafil M 2125 CS (Gattefosse) 4 PEG-6 almond oilLabrafil M 1966 CS (Gattefosse) 4 PEG-6 apricot kernel oil Labrafil M1944 CS (Gattefosse) 4 PEG-6 olive oil Labrafil M 1980 CS (Gattefosse) 4PEG-6 peanut oil Labrafil M 1969 CS (Gattefosse) 4 PEG-6 hydrogenatedpalm Labrafil M 2130 BS (Gattefosse) 4 PEG-6 palm kernel oil Labrafil M2130 CS (Gattefosse) 4 PEG-6 triolein Labrafil M 2735 CS (Gattefosse) 4PEG-8 corn oil Labrafil WL 2609 BS (Gattefosse) 6-7 PEG-20 cornglycerides Crovol M40 (Croda) 10 PEG-20 almond glycerides Crovo A40(Croda) 10 PEG-25 trioleate TAGAT TO (Goldschmidt) 11 PEG-40 palm kerneloil Crovol PK-70 >10 PEG-60 corn glycerides Crovol M70 (Croda) 15 PEG-60almond glycerides Crovol A70 (Croda) 15 PEG-4 caprylic/capric LabrafacHydro (Gattefosse) 4-5 PEG-8 caprylic/capric Labrasol (Gattefosse),Labrafac CM 10 (Gattefosse) >10 glycerides PEG-6 caprylic/capricSOFTIGEN 767 (Huls), Glycerox 767 (Croda) 19 glycerides Lauroylmacrogol-32 glyceride GELUCIRE 44/14 (Gattefosse) 14 Stearoyl macrogolglyceride GELUCIRE 50/13 (Gattefosse) 13 Mono, di, tri, tetra esters ofSorbitolGlyceride (Gattefosse) <10 vegetable oils and sorbitolPentaerythrityl tetraisostearate Crodamol PTIS (Croda) <10Pentaerythrityl distearate Albunol DS (Taiwan Surf.) >10 Pentaerythrityltetraoleate Liponate PO-4 (Lipo Chem.) >10 Pentaerythrityl tetrastearateLiponate PS-4 (Lipo Chem.) <10 Pentaerythrityl tetracaprylate/ LiponatePE-810 (Lipo Chem.), <10 tetracaprate Crodamol PTC (Croda)Pentaerythrityl tetraoctanoate Nikkol Pentarate 408 (Nikko)

Also included as oils in this category of surfactants are oil-solublevitamins, such as vitamins A, D, E, K, etc. Thus, derivatives of thesevitamins, such as tocopheryl PEG-100 succinate (TPGS, available fromEastman), are also suitable surfactants.

6. Polyglycerized Fatty Acids

Polyglycerol esters of fatty acids are also suitable surfactants for thepresent invention. Among the polyglyceryl fatty acid esters, preferredhydrophobic surfactants include polyglyceryl oleate (Plurol Oleique),polyglyceryl-2 dioleate (Nikkol DGDO), and polyglyceryl-10 trioleate.Preferred hydrophilic surfactants include polyglyceryl-10 laurate(Nikkol Decaglyn 1-L), polyglyceryl-10 oleate (Nikkol Decaglyn 1-0), andpolyglyceryl-10 mono, dioleate (Caprool PEG 860). Polyglycerylpolyricinoleates (Polymuls) are also preferred hydrophilic andhydrophobic surfactants. Examples of suitable polyglyceryl esters areshown in Table 6.

TABLE 6 Polyglycerized Fatty Acids Compound Commercial Product(Supplier) HLB Polyglyceryl-2 stearate Nikkol DGMS (Nikko) 5-7Polyglyceryl-2 oleate Nikkol DGMO (Nikko) 5-7 Polyglyceryl-2 isostearateNikkol DGMIS (Nikko) 5-7 Polyglyceryl-3 oleate Caprol 3GO (ABITEC),Drewpol 3-1-O (Stepan) 6.5 Polyglyceryl-4 oleate Nikkol Tetraglyn 1-O(Nikko) 5-7 Polyglyceryl-4 stearate Nikkol Tetraglyn 1-S (Nikko) 5-6Polyglyceryl-6 oleate Drewpol 6-1-O (Stepan), Nikkol 9 Hexaglyn 1-O(Nikko) Polyglyceryl-10 laurate Nikkol Decaglyn 1-L (Nikko) 15Polyglyceryl-10 oleate Nikkol Decaglyn 1-O (Nikko) 14 Polyglyceryl-10stearate Nikkol Decaglyn 1-S (Nikko) 12 Polyglyceryl-6 ricinoleateNikkol Hexaglyn PR-15 (Nikko) >8 Polyglyceryl-10 linoleate NikkolDecaglyn 1-LN (Nikko) 12 Polyglyceryl-6 pentaoleate Nikkol Hexaglyn 5-O(Nikko) <10 Polyglyceryl-3 dioleate Cremophor GO32 (BASF) <10Polyglyceryl-3 distearate Cremophor GS32 (BASF) <10 Polyglyceryl-4pentaoleate Nikkol Tetraglyn 5-O (Nikko) <10 Polyglyceryl-6 dioleateCaprol 6G20 (ABITEC); Hodag PGO-62, 8.5 (Calgene) PLUROL OLEIQUE CC 497(Gattefosse) Polyglyceryl-2 dioleate Nikkol DGDO (Nikko) 7Polyglyceryl-10 trioleate Nikkol Decaglyn 3-O (Nikko) 7 Polyglyceryl-10pentaoleate Nikkol Decaglyn 5-O (Nikko) 3.5 Polyglyceryl-10 septaoleateNikkol Decaglyn 7-O (Nikko) 3 Polyglyceryl-10 tetraoleate Caprol 10G40(ABITEC; Hodag PGO-62 (CALGENE), Drewpol 10-4-O (Stepan) Polyglyceryl-10decaisostearate Nikkol Decaglyn 10-IS (Nikko) <10 Polyglyceryl-101decaoleate Drewpol 10-10-O (Stepan), Caprol 10G10O 3.5 (ABITEC), NikkolDecaglyn 10-O Polyglyceryl-10 mono, dioleate Caprol PGE 860 (ABITEC) 11Polyglyceryl polyricinoleate Polymuls (Henkel)  3-20

7. Propylene Glycol Fatty Acid Esters

Esters of propylene glycol and fatty acids are suitable surfactants foruse in the present invention. In this surfactant class, preferredhydrophobic surfactants include propylene glycol monolaurate(Lauroglycol FCC), propylene glycol ricinoleate (Propymuls), propyleneglycol monooleate (Myverol P-06), propylene glycol dicaprylate/dicaprate(Captex 200), and propylene glycol dioctanoate (Captex 800). Examples ofsurfactants of this class are given in Table 7.

TABLE 7 Propylene Glycol Fatty Acid Esters Compound Commercial Product(Supplier) HLB Propylene glycol monocaprylate Capryol 90 (Gattefosse),<10 Nikkol Sefsol 218 (Nikko) Propylene glycol monolaurate Lauroglycol90 (Gattefosse), Lauroglycol <10 FCC (Gattefosse) Propylene glycololeate Lutrol OP2000 (BASF) <10 Propylene glycol myristate Mirpyl <10Propylene glycol monostearate ADM PGME-03 (ADM), LIPO PGMS 3-4 (LipoChem.), Aldo PGHMS (Lonza) Propylene glycol hydroxyl stearate <10Propylene glycol ricinoleate PROPYMULS (Henkel) <10 Propylene glycolisostearate <10 Propylene glycol monooleate Myverof P-06 (Eastman) <10Propylene glycol dicaprylate/ Captex 200 (ABITEC), Miglyol 840 (Huls),<6 dicaprate Neobee M-20 (Stepan) Propylene glycol dioctanoate Captex800 (ABITEC) <6 Propylene glycol caprylate/caprate LABRAFAC PG(Gattefosse) >6 Propylene glycol dilaurate >6 Propylene glycoldistearate Kessco PGDS (Stepan) >6 Propylene glycol dicaprylate NikkolSefsol 228 (Nikko) >6 Propylene glycol dicaprate Nikkol PDD (Nikko)

8. Mixtures of Propylene Glycol Esters-Glycerol Esters

In general, mixtures of surfactants are also suitable for use in thepresent invention. In particular, mixtures of propylene glycol fattyacid esters and glycerol fatty acid esters are suitable and arecommercially available. One preferred mixture is composed of the oleicacid esters of propylene glycol and glycerol (Arlacel 186). Examples orthese surfactants are shown in Table 8.

TABLE 8 7/26 Glycerol/Propylene Glycol Fatty Acid Esters CompoundCommercial Product (Supplier) HLB Oleic ATMOS 300, ARLACEL 186 (ICI) 3-4Stearic ATMOS 150 3-4

9. Mono-Dialycerides

A particularly important class of surfactants is the class of mono- anddiglycerides. These surfactants are generally hydrophobic. Preferredhydrophobic surfactants in this class of compounds include glycerylmonooleate (Peceol), glyceryl ricinoleate, glyceryl laurate, glyceryldilaurate (Capmul GDL), glyceryl dioleate (Capmul GDO), glycerylmono/dioleate (Capmul GMO-K), glyceryl caprylate/caprate (Capmul MCM),caprylic acid mono/diglycerides (Imwitor 988), and mono- anddiacetylated monoglycerides (Myvacet 9-45). Examples of thesesurfactants are given in Table 9.

TABLE 9 Mono- and Diglyceride Surfactants Compound Commercial Product(Supplier) HLB Monopalmitolein (C16:1) (Larodan) <10 Monoelaidin (C18:1)(Larodan) <10 Monocaproin (C6) (Larodan) <10 Monocaprylin (Larodan) <10Monocaprin (Larodan) <10 Monolaurin (Larodan) <10 Glyceryl monomyristate(C14) Nikkol MGM (Nikko) 3-4 Glyceryl monooleate (C18:1) PECEOL(Gattefosse), Hodag GMO-D, 3-4 Nikkol MGO (Nikko) Glyceryl monooleateRYLO series (Danisco, DIMODAN series (Danisco), EMULDAN (Danisco), ALDOMO FH (Lonza), Kessco GMO (Stepan), MONOMULS series (Henkel), TEGIN O,DREWMULSE GMO (Stepan), Atlas G-695 (ICI), GMOrphic 80 (Eastman), ADMDMG-40, 70, and 100 (ADM), Myverol (Eastman) Glycerylmono-oleate/linoleate OLICINI (Gattefosse) 3-4 Glyceryl monolineateMaisine (Gattefosse), MYVEROL 18-92, Myverol 18-06 (Eastman) Glycerylricinoleate Softigen 701 (Huls), HODAG GMR-D 6 (Calgene), ALDO MR(Lonza) Glyceryl monolaurate ALDO MLD (Lonza), Hodag GML 6.8 (Calgene)Glyceryl monopalmitate Emalex GMS-P (Nihon) 4 Glyceryl monostearateCapmul GMS (ABITEC), Myvaplex, 5-9 IMWITOR 191 (Huls), CUTINA GMS, AldoMS (Lonza), Nikkol MGS series (Nikko) Glyceryl mono-, dioleate CapmulGMO-K (ABITEC) <10 Glyceryl palmitic/stearic CUTINA MD-A, ESTAGEL-G18<10 Glyceryl acetate Lamegin EE (Gunau GmbH) <10 Glyceryl laurateImwitor 312 (Huls), Monoluls 90-45 4 (Grunau GmbH), Aldo MLD (Lonza)Glyceryl citrate/lactate/oleate/ Imwitor 375 (Huls) <10 linoleateGlyceryl caprylate Imwitor 308 (Huls), Capmul MCMS 5-6 (ABITEC) Glycerylcaprylate/caprate Capmul MCM (ABITEC) 5-6 Caprylic acid mono,diglycerides Imwitor 988 (Huls) 5-6 Caprylic/capric glycerides Imwitor742 (Huls) <10 Mono- and monoglycerides Myvacet 9-45, Myvacet 9-40,Myvacet 9-08 3.8-4   (Eastman), Lamegin (Grunau) Glyceryl monostearateAldo MS, Arlacel 129 (ICI), LIPO GMS 4.4 (Lipo Chem.), Imwitor 191(Huls), Myvaplex (Eastman) Lactic acid esters of mono, LAMEGIN GLP(Henkel) <10 diclycerides Dicaproin (C6) (Larodan) <10 Dacaprin (C10)(Larodan) <10 Dioctanoin (C8) (Larodan) <10 Dimyristin (C14) (Larodan)<10 Dipalmitin (C16) (Larodan) <10 Distearin (Larodan) <10 Glyceryldilaurate (C12) Capmul GDL (ABITEC) 3-4 Glyceryl dioleate Capmul GDO(ABITEC) 3-4 Glyceryl esters of fatty acids GELUCIRE 39/01 (Gattefosse),GELUCIRE 6 43/01 (Gattefosse), GELUCIRE 37/06 (Gattefosse) Dipalmitolein(C16:1) (Larodan) <10 1,2 and 1,3-diolein (C18:1) (Larodan) <10Dielaidin (C18:1) (Larodan) <10 Dilinolein (C18:2) (Larodan) <10

10. Sterol and Sterol Derivatives

Sterols and derivatives of sterols are suitable surfactants for use inthe present invention. These surfactants can be hydrophilic orhydrophobic. Preferred derivatives include the polyethylene glycolderivatives. A preferred hydrophobic surfactant in this class ischolesterol. A preferred hydrophilic surfactant in this class is PEG-24cholesterol ether (Solulan C-24). Examples of surfactants of this classare shown in Table 10.

TABLE 10 Sterol and Sterol Derivative Surfactant Compound CommercialProduct (Supplier) HLB Cholesterol, sitosterol, Ianosterol <10 PEG-24cholesterol ether Solulan C-24 (Amerchol) >10 PEG-30 cholestanol NikkolDHC (Nikko) >10 Phytosterol GENEROL series (Henkel) <10 PEG-25 phytosterol Nikkol BPSH-25 (Nikko) >10 PEG-5 soya sterol Nikkol BPS-5 (Nikko)<10 PEG-10 soya sterol Nikkol BPS-10 (Nikko) <10 PEG-20 soya sterolNikkol BPS-20 (Nikko) <10 PEG-30 soya sterol Nikkol BPS-30 (Nikko) >10

11. Polyethylene Glycol Sorbitan Fatty Acid Esters

A variety of PEG-sorbitan fatty acid esters are available and aresuitable for use as surfactants in the present invention. In general,these surfactants are hydrophilic, although several hydrophobicsurfactants of this class can be used. Among the PEG-sorbitan fatty acidesters, preferred hydrophilic surfactants include PEG-20 sorbitanmonolaurate (Tween-20), PEG-20 sorbitan monostearate (Tween-60), andPEG-20 sorbitan monooleate (Twee-80). Examples of these surfactants areshown in Table 11.

TABLE 11 PEG-Sorbitan Fatty Acid Esters Compound Commercial Product(Supplier) HLB PEG-10 sorbitan laurate Liposorb L-10 (Lipo Chem.) >10PEG-20 sorbitan monolaurate Tween-20 (Atlas/ICI), Crillet 1 (Croda),DACOL 17 MLS 20 (Condea) PEG-4 sorbitan monolaurate Tween-21(Atlas/ICI), Crillet 11 (Croda) 13 PEG-80 sorbitan monolaurate HodagPSML-80 (Calgene); T-Maz 28 >10 PEG-6 sorbitan Nikkol GL-1 (Nikko) 16PEG-20 sorbitan monopalmitate Tween 40 (Atlas/ICI), Crillet 2 (Croda) 16PEG-20 sorbitan monostearate Tween-60 (Atlas/ICI), Crillet 3 (Croda) 15PEGA sorbitan monostearate Tween-61 (Atlas/ICI), Crillet 31 (Croda) 9.6PEG-8 sorbitan monostearate DACOL MSS (Condea) >10 PEG-6 sorbitanmonostearate Nikkol TS106 (Nikko) 11 PEG-20 sorbitan tristearateTween-65 (Atlas/ICI), Crillet 35 (Croda) 11 PEG-6 sorbitan tetrastearateNikkol GS-6 (Nikko) 3 PEG-60 sorbitan tetrastearate Nikkol GS-460(Nikko) 13 PEG-5 sorbitan monooleate Tween-81 (Atlas/ICI), Crillet 41(Croda) 10 PEG-6 sorbitan monooleate Nikkol TO-106 (Nikko) 10 PEG-20sorbitan monooleate Tween-80 (Atlas/ICI), Crillet 4 (Croda) 15 PEG-40sorbitan oleate Emalex ET 8040 (Nihon Emulsion) 18 PEG-20 sorbitantrioleate Tween-85 (Atlas/ICI), Crillet 45 (Croda) 11 PEG-6 sorbitantetraoleate Nikkol GO-4 (Nikko) 8.5 PEG-30 sorbitan tetraoleate NikkolG-430 (Nikko) 12 PEG-40 sorbitan tetraoleate Nikkol GO-440 (Nikko) 13PEG-20 sorbitan monoisostearate Tween-120 (Atlas/ICI), Crillet 6(Croda) >10 PEG sorbitol hexaoleate Atlas G-1086 (ICI) 10 PEG-6 sorbitolhexastearate Nikkol GS-6 (Nikko) 3

12. Polyethylene Glycol Alkyl Ethers

Ethers of polyethylene glycol and alkyl alcohols are suitablesurfactants for use in the present invention. Preferred hydrophobicethers include PEG-3 oleyl ether (Volpo 3) and PEG-4 lauryl ether (Brij30). Examples of these surfactants are shown in Table 12.

TABLE 12 Polyethylene Glycol Alkyl Ethers Commercial Compound Product(Supplier) HLB PEG-2 oleyl ether, oleth-2 Brij 92/93 (Atlas/ICI) 4.9PEG-3 oleyl ether, oleth-3 Volpo 3 (Croda) <10 PEG-5 oleyl ether,oleth-5 Volpo 5 (Croda) <10 PEG-10 oleyl ether, oleth-10 Volpo 10(Croda), 12 Brij 96/97 (Atlas/ICI) PEG-20 oleyl ether, oleth-20 Volpo 20(Croda), 15 Brij 98/99 (Atlas/ICI) PEG-4 lauryl ether, laureth-4 Brij 30(Atlas/ICI) 9.7 PEG-9 lauryl ether >10 PEG-23 lauryl ether, Brij 35(Atlas/ICI) 17 laureth-23 PEG-2 cetyl ether Brij 52 (ICI) 5.3 PEG-10cetyl ether Brij 56 (ICI) 13 PEG-20 cetyl ether Brij 58 (ICI) 16 PEG-2stearyl ether Brij 72 (ICI) 4.9 PEG-10 stearyl ether Brij 76 (ICI) 12PEG-20 stearyl ether Brij 78 (ICI) 1 PEG-100 stearyl ether Brij 100(ICI) >10

13. Sugar Esters

Esters of sugar are suitable surfactants for using the presentinvention. Preferred hydrophilic surfactants in this class includesucrose monopalmitate and sucrose monolaurate. Examples of suchsurfactants are shown in Table 13.

TABLE 13 Sugar Ester Surfactants Compound Commercial Product (Supplier)HLB Sucrose distearate SUCRO ESTER 7 (Gattefosse), 3 Crodesta F-10(Croda) Sucrose distearate/ SUCRO ESTER 11 (Gattefosse), 12 monostearateCrodesta F-110 (Croda) Sucrose dipalmitate 7.4 Sucrose monostearateCrodesta F-160 (Croda) 15 Sucrose mono-palmitate SUCRO ESTER 15(Gattefosse) >10 Sucrose monolaurate Saccharose monolaurate 15 1695(Mitsubishi-Kasei)

14. Polyethylene Glycol Alkyl Phenols

Several hydrophilic PEG-alkyl phenol surfactants are available, and aresuitable for use in the present invention. Examples of these surfactantsare shown in Table 14.

TABLE 14 Polyethylene Glycol Alkyl Phenol Surfactants CompoundCommercial Product (Supplier) HLB PEG-10-100 Triton X series (Rohm &Haas), Igepal CA >10 nonyl phenol series (GAF, USA), Antarox CA series(GAF, UK) PEG-15-100 Triton N-series (Rohm & Haas), Igepal CO series >10octyl phenol (GAF, USA), Antarox CO series (GAF, UK) ether

15. Polyoxyethylene-Polyoxypropylene Block Copolymers

The POE-POP block copolymers are a unique class of polymericsurfactants. The unique structure of the surfactants, with hydrophilicPOE and hydrophobic POP moieties in well-defined ratios and positions,provides a wide variety of surfactants suitable for use in the presentinvention. These surfactants are available under various trade names,including Synperonic PE series (ICI); Pluronic series (BASF), Emkalyx,Lutrol (BASF), Supronic, Monolan, Pluracare, and Plurodac. The genericterm for these polymers is “poloxamer” (CAS 9003-11-6). These polymershave the formula: HO(C₂H₄O)_(a)(C₃H₆O)_(b)(C₂H₄O)_(a)H where “a” and “b”denote the number of polyoxyethylene and polyoxypropylene units,respectively. Preferred hydrophilic surfactants of this class includePoloxamers, 108, 188, 217, 238, 288, 338, and 407. Preferred hydrophobicsurfactants in this class include Poloxamers 124, 182, 183, 212, 331,and 335. Examples of suitable surfactants of this class are shown inTable 15. Since the compounds are widely available, commercial sourcesare not listed in the Table. The compounds are listed by generic name,with the corresponding “a” and “b” values.

TABLE 15 POE-POP Block Copolymers a, b values in CompoundHO(C₂H₄O)_(a)(C₃H₆O)_(b)(C₂H₄O)_(a)H HLB Poloxamer 105 a = 11 b = 16 8Poloxamer 108 a = 46 b = 16 >10 Poloxamer 122 a = 5 b = 21 3 Poloxamer123 a = 7 b = 21 7 Poloxamer 123 a = 11 b = 21 >7 Poloxamer 181 a = 3 b= 30 Poloxamer 182 a = 8 b = 30 2 Poloxamer 183 a = 10 b = 30 Poloxamer184 a = 13 b = 30 Poloxamer 185 a = 19 b = 30 Poloxamer 188 a = 75 b =30 29 Poloxamer 212 a = 8 b = 35 Poloxamer 215 a = 24 b = 35 Poloxamer217 a = 52 b = 35 Poloxamer 231 a = 16 b = 39 Poloxamer 234 a = 22 b =39 Poloxamer 235 a = 27 b = 39 Poloxamer 237 a = 62 b = 39 24 Poloxamer238 a = 97 b = 39 Poloxamer 282 a = 10 b = 47 Poloxamer 284 a = 21 b =47 Poloxamer 288 a = 122 b = 47 >10 Poloxamer 331 a = 7 b = 54 0.5Poloxamer 333 a = 20 b = 54 Poloxamer 334 a = 31 b = 54 Poloxamer 335 a= 38 b = 54 Poloxamer 338 a = 128 b = 54 Poloxamer 401 a = 6 b = 67Poloxamer 402 a = 13 b = 67 Poloxamer 403 a = 21 b = 67 Poloxamer 407 a= 98 b = 67

16. Sorbitan Fatty Acid Esters

Sorbitan esters of fatty acids are suitable surfactants for use in thepresent invention. Among these esters, preferred hydrophobic surfactantsinclude sorbitan monolaurate (Arlacel 20), sorbitan monopalmitate(Span-40), sorbitan monooleate (Span-80), sorbitan monostearate, andsorbitan tristearate. Examples of these surfactants are shown in Table16.

TABLE 16 Sorbitan Fatty Acid Esters Surfactants Compound CommercialProduct (Supplier) HLB Sorbitan monolaurate Span-20 (Atlas/ICI), Crill 1(Croda), Arlacel 20 8.6 (ICI) Sorbitan monopalmitate Span-40(Atlas/ICI), Crill 2 (Croda), Nikkol 6.7 SP-10 (Nikko) Sorbitanmonooleate Span-80 (Atlas/ICI), Crill 4 (Croda), Crill 50 4.3 (Croda)Sorbitan monostearate Span-60 (Atlas/ICI), Crill 3 (Croda), Nikkol 4.7SS-10 (Nikko) Sorbitan trioleate Span-85 (Atlas/ICI), Crill 45 (Croda),Nikkol 4.3 SO-30 (Nikko) Sorbitan sesquioleate Arlacel-C (ICI), Crill 43(Croda), Nikkol SO-15 3.7 (Nikko) Sorbitan tristearate Span-65(Atlas/ICI) Crill 35 (Croda), Nikkol SS-30 (Nikko) 2.1 Sorbitanmonoisostearate Crill 6 (Croda), Nikkol SI-10 (Nikko) 4.7 Sorbitansesquistearate Nikkol SS-15 (Nikko) 4.2

17. Lower Alcohol Fatty Acid Esters

Esters of lower alcohols (C₂ to C₄) and fatty acids (C₈ to C₁₈) aresuitable surfactants for use in the present invention. Among theseesters, preferred hydrophobic surfactants include ethyl oleate (CrodamolEO), isopropyl myristate (Crodamol IPM), and isopropyl palmitate(Crodamol IPP). Examples of these surfactants are shown in Table 17.

TABLE 17 Sorbitan Fatty Acid Esters Surfactants Compound CommercialProduct (Supplier) HLB Ethyl oleate Crodamol EO (Croda), Nikkol EOO(Nikko) <10 Isopropyl myristate Crodamol IPM (Croda) <10 Isopropylpalmitate Crodamol IPP (Croda) <10 Ethyl linoleate Nikkol VF-E (Nikko)<10 Isopropyl linoleate Nikkol VF-IP (Nikko) <10

18. Ionic Surfactants

Ionic surfactants, including cationic, anionic and zwitterionicsurfactants, are suitable hydrophilic surfactants for use in the presentinvention. Preferred anionic surfactants include fatty acid salts andbile salts. Specifically, preferred ionic surfactants include sodiumoleate, sodium lauryl sulfate, sodium lauryl sarcosinate, sodium dioctylsulfosuccinate, sodium cholate, and sodium taurocholate. Examples ofsuch surfactants are shown in Table 18 below. For simplicity, typicalcounterions are shown in the entries in the Table. It will beappreciated by one skilled in the art; however, that any bioacceptablecounterion may be used. For example, although the fatty acids are shownas sodium salts, other cation counterions can also be used, such asalkali metal cations or ammonium. Unlike typical non-ionic surfactants,these ionic surfactants are generally available as pure compounds,rather than commercial (proprietary) mixtures. Because these compoundsare readily available from a variety of commercial suppliers, such asAldrich Sigma, and the like, commercial sources are not generally listedin the Table.

TABLE 18 Ionic Surfactants Compound HLB FATTY ACID SALTS >10 Sodiumcaproate Sodium caprylate Sodium caprate Sodium laurate Sodium myristateSodium myristolate Sodium palmitate Sodium palmitoleate Sodium oleate 18Sodium ricinoleate Sodium linoleate Sodium linolenate Sodium stearateSodium lauryl sulfate (dodecyl) 40 Sodium tetradecyl sulfate Sodiumlauryl sarcosinate Sodium dioctyl sulfosuccinate (sodium docusate(Cytec)) BILE SALTS >10 Sodium cholate Sodium taurocholate Sodiumglycocholate Sodium deoxycholate Sodium taurodeoxycholate Sodiumglycodeoxycholate Sodium ursodeoxycholate Sodium chenodeoxycholateSodium taurochenodeoxycholate Sodium glycol cheno deoxycholate Sodiumcholylsarcosinate Sodium N-methyl taurocholate PHOSPHOLIPIDS Egg/Soylecithin (Epikuron (Lucas Meyer), Ovothin (Lucas Meyer)) Lyso egg/soylecithin Hydroxylated lecithin Lysophosophatidylcholine CardiolipinSphingomyelin Phosphatidylcholine Phosphatidyl ethanolamine Phosphatidicacid Phophatidyl glycerol Phosphatidyl serine PHOSPHORIC ACID ESTERSDiethanolammonium polyoxyethylene-10 oleyl ether phosphateEsterification products of fatty alcohols or fatty alcohol ethoxylateswith phosphoric acid or anhydride CARBOXYLATES Ether carboxylates (byoxidation of terminal OH group of fatty alcohol ethoxylates)Succinylated monoglycerides (LAMEGIN ZE (Henkel)) Sodium stearylfumarate Stearoyl propylene glycol hydrogen succinate Mono/diacetylatedtartaric acid esters of mono- and diglycerides Citric acid esters ofmono-, diglycerides Glyceryl-lacto esters of fatty acids (CFR ref.172.852) Acyl lactylates lactylic esters of fatty acids calcium/sodiumstearoyl-2-lactylate calcium/sodium stearoyl lactylate Alginate saltsPropylene glycol alginate SULFATES AND SULFONATES Ethoxylated alkylsulfates Alkyl benzene sulfones- olefin sulfonates Acyl isethionatesAcyl taurates Alkyl glyceryl ether sulfonates Octyl sulfosuccinatedisodium Disodium undecyclenamideo-MEA-sulfosuccinate CATIONICSurfactants >10 Hexadecyl triammonium bromide Decyl trimethyl ammoniumbromide Cetyl trimethyl ammonium bromide Dodecyl ammonium chloride Alkylbenzyldimenthylammonium salts Diisobutyl phenoxyethoxydimethylbenzylammonium salts Alkylpyridinium salts Betaines (trialkylglycine):Lauryl betaine (N-lauryl, N, N-dimenthylglycine) Ethoxylated amines:Polyoxyethylene-15 coconut amine

Examples of additional suitable solubilizer include: alcohols andpolyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethyleneglycol, propylene glycol, butanediols and isomers thereof, glycerol,pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide,polyethylene glycol, polypropylene glycol, polyvinylalcohol,hydroxypropyl methylcellulose and other cellulose derivatives,cyclodextrins and cyclodextrin derivatives; ethers of polyethyleneglycols having an average molecular weight of about 200 to about 6000,such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, availablecommercially from BASF under the trade name Tetraglycol) or methoxy PEG(Union Carbide); amides, such as 2-pyrrolidone, 2-piperidone,caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone,N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, andpolyvinypyrrolidone; esters, such as ethyl propionate, tributylcitrate,acetyl triethylcitrate, acetyl tributyl citrate, triethylcitrate, ethyloleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycolmonoacetate, propylene glycol diacetate, caprolactone and isomersthereof, valerolactone and isomers thereof, butyrolactone and isomersthereof; and other solubilizers known in the art, such as dimethylacetamide, dimethyl isosorbide (Arlasolve DMI (ICI)),N-methylpyrrolidones (Pharmasolve (ISP)), monooctanoin, diethyleneglycol nonoethyl ether (available from Gattefosse under the trade nameTranscutol), and water. Mixtures of solubilizers are also within thescope of the invention.

Except as indicated, compounds mentioned herein are readily availablefrom standard commercial sources.

Particularly suitable water miscible solvents include, by way of exampleand without limitation, ethanol or iso-propyl alcohol, poly(ethyleneglycol). Particularly suitable emulsifying agents include, by way ofexample and without limitation, is glycerol monooleate, vitamin E TPGS,Gelucire, Cremophor, Labrafil, poloxamer and Labrasol. Particularlysuitable water immiscible solvents include, by way of example andwithout limitation, medium chain triglycerides and oleic acid.Particularly suitable antioxidants include, by way of example andwithout limitation, vitamin E, BHT, or vitamin C palmitate.

Selection of excipients suitable for use in the solubilizer wasconducted according to Example 4, Method A. Some of the suitableexcipients include triglycerides of unsaturated fatty acids. Thesecompounds are susceptible to oxidation, so an antioxidant is preferablyincluded therewith in a composition of the invention. It is noted thateven though a solubilizer (which is an excipient) might not be suitablefor individually solubilizing the SCF extract, such an excipient can beused in a composition as a mixture with one or more other excipientsthat solubilize the SCF extract.

A pharmaceutical liquid composition of the invention can be clear or asuspension. Clarity of the liquid composition was determined visuallywith the unaided eye or with a microscope using the method of Example 5.The clear liquid composition is visually clear to the unaided eye, as itwill contain less than 5%, less than 3% or less than 1% by wt. ofsuspended solids based upon the total weight of the composition.Specific embodiments of the invention include a pharmaceutical clearliquid composition that can be used as a fill composition in a capsulethereby forming a liquid filled capsule formulation. The clear liquidcomposition is made by mixing the SCF extracts with a solubilizer of theinvention, optionally in the presence of heat, wherein the solubilizeris present in an amount sufficient to dissolve the extract.

Exemplary liquid compositions of the invention are described in Example3. The composition of Example 3, Method A is a cremophor-based drugdelivery system. The composition of Example 3, Method B is a GMO(glycerol monooleate)/cremophor-based drug delivery system. Thecomposition of Example 3, Method C is a labrasol-based micelle formingsystem. Each of these formulations includes an antioxidant since thesurfactant excipient contains unsaturated fatty acid, which is asolubilizing agent. They also include ethanol as a water soluble(miscible) solvent.

As used herein, the term “micelle forming system” refers to acomposition that forms a micellar dispersion or emulsion when placed inan aqueous medium. As used herein, the term “self-emulsifying system”refers to a composition that forms an emulsion when placed in an aqueousmedium.

The composition of Example 3, Method D is a Vitamin E TPGS based drugdelivery system.

The dissolution properties of the formulation, when placed in an aqueousmedium, were evaluated according to Example 4, Method C. When thecomposition of Example 3, Method A was placed in phosphate buffer (pH6.8), micelles formed and the composition dissolved in the buffer. Whenthe composition of Example 3, Method B was placed in phosphate buffer(pH 6.8), the composition dispersed in the buffer. When the liquidcomposition of Example 3, Method C was placed in phosphate buffer (pH6.8), the formation of fine particles in the buffer was observed. Whenthe composition of Example 3, Method D was placed in phosphate buffer(pH 6.8), a micellular dispersion was formed.

If desired the liquid composition can be sterilized by: 1) sterilefiltering the fill composition through a filtration medium wherein thepore size is about 0.22 μm or smaller; 2) irradiating the fillcomposition; 3) treating the fill composition with ethylene oxide; 4)purging the fill composition with an inert gas to reduce the amount ofdissolved oxygen therein; and/or 5) heating the fill composition.

A capsule formulation comprises a shell, a pharmaceutical liquidcomposition filling, and optionally, an enteric coat. A capsuleaccording to the invention will have a storage shelf-life of no lessthan one week, three weeks, one month, three months, six months, or oneyear. For example, for a capsule having a shelf life of at least sixmonths, the shell of the capsule will not fail storage stability testsfor a storage period of at least six months. The criteria for acceptableshelf-life are set as needed according to a given capsule product andits storage stability requirements. It should be noted that a shelf-lifeof as little as one week is suitable for products that are compounded bya pharmacist and sold to customers of a pharmacy.

The loading or filling of a liquid composition into a capsule can beachieved by any known method for preparing liquid, gel, semi-solid orsolid melt filled capsules. In particular, the methods described by R.P. Scherer company, Alza or MW Encap Ltd. can be used. One exemplarymethod is described by Bowtle (Pharmaceutical Technology Europe (1998),10 (10), 84, 86, 88-90.

The term “shell” as used herein is taken to mean the shell of a capsuledosage form or the encasement or encapsulation material used toencapsulate fill compositions made from the particles. Any materialsuitable for use in forming a capsule shell or in encapsulating anothercomposition can be used according to the invention.

The shell can be hard or soft and any materials suitable for preparingsuch shells can be used in the capsule of the invention. Materialssuitable for the preparation of the capsule shell include soft gelatin,hard gelatin, hydroxypropyl methylcellulose, starch, animal gelatin,agar, fish (piscine) gelatin or a combination thereof. Other suitablematerials include: polyinyl alcohol/polyvinyl acetate copolymer (U.S.Pat. No. 3,300,546); a blend of hydroxybutyl methylcellulose andhydroxypropyl methylcellulose (U.S. Pat. No. 4,765,916); polyvinylacetate (U.S. Pats. No. 2,560,649, No. 3,346,502); water-soluble gelatin(U.S. Pat. No. 3,525,426); polyvinyl alcohol (U.S. Pat. No. 3,528,921,No. 3,534,851, No. 3,556,765, No. 3,634,260, No. 3,671,439, No.3,706,670, No. 3,857,195, No. 3,877,928, No. 4,367,156, No. 4,747,976,No. 5,270,054); polymers derived from such monomers as vinyl chloride,vinyl alcohol, vinyl pyrrolidone, furan, acrylonitrile, vinyl acetate,methyl acrylate, methyl methacrylate, styrene, vinyl ethyl ether, vinylpropyl ether, acrylamide, ethylene, propylene, acrylic acid, methacrylicacid, maleic anhydride, salts of any of the aforementioned acids andmixtures thereof; polyvinyl chloride; polypropylene; acrylic/maleiccopolymers; sodium polyacrylate; polyvinyl pyrrolidone; glucomannan andoptionally another natural polysaccharide with a polyhydric alcohol suchas glycerin (U.S. Pat. No. 4,851,394); plastic andpolylactide/polyglycolide (Elanco Animal Health Co.); HPMC (ShionogiQualicaps Co. Ltd (Nara Japan); SUHEUNG CAPSULES CO. LTD. (KYUNGGI-DO,KOREA) and Capsugel); or a combination thereof. Essentially any materialknown to those of ordinary skill in the art as being for the preparationof capsule shell can be used in a capsule according to the invention.Suitable starch capsules can be made and used according to Vilivalam etal. (Pharmaceutical Science & Technology Today (2000), 3 (2), 64-69). Achitosan capsule for colonic delivery can be made and used according toYamamoto (Kobunshi (1999), 48 (8), 595) or Tozaki et al. (Drug DeliverySystem (1997), 12 (5), 311-320). Other suitable shell materials aredisclosed in U.S. Patent Application Publication No. 2002/0081331 to R.P. Scherer Technologies Inc. (Cardinal Health, Inc.), which disclosesfilm-forming compositions comprising modified starches andiota-carrageenan.

The capsule of the invention can also be coated with an enteric coat todelay release of its contents until it is downstream from the gastricregion following oral administration or until it is exposed to anaqueous medium having a pH of at least about 5. An enteric coatedcapsule can be adapted to release the liquid composition in theduodenum, jejunum, ileum, small intestine or large intestine.

The enteric coat (delayed release coat) is exterior to and surrounds(encloses or envelopes) the capsule shell. The coating is insoluble inthe fluid of a first environment of use, such as gastric juices, acidicfluids, and soluble or erodible in the fluid of a second environment ofuse, such as intestinal juices, substantially pH neutral or basicfluids, or mildly acidic (pH of 5 or greater) fluids. Many polymericmaterials are known to possess these various solubility properties andcan be included in the enteric coat. Such other polymeric materialsinclude, by way of example and without limitation, cellulose acetatephthalate (CAP), cellulose acetate trimelletate (CAT), poly(vinylacetate) phthalate (PVAP), hydroxypropylmethylcellulose phthalate (HP),poly(methacrylate ethylacrylate) (1:1) copolymer (MA-EA),poly(methacrylate methylmethacrylate) (1:1) copolymer (MA-MMA),poly(methacrylate methylmethacrylate) (1:2) copolymer, Eudragit L-30-D™(MA-EA, 1:1), Eudragit L-100-55™ (MA-EA, 1:1),hydroxypropylmethylcellulose acetate succinate (HPMCAS), Coateric™(PVAP), Aquateric™ (CAP), AQOAT™ (HPMCAS) and combinations thereof.

When the enteric coat is intended to be dissolved, eroded or becomedetached from the capsule in the colon, materials such ashydroxypropylcellulose, microcrystalline cellulose (MCC, Avicel™ fromFMC Corp.), poly (ethylene—vinyl acetate) (60:40) copolymer (EVAC fromAldrich Chemical Co.), 2-hydroxyethylmethacrylate (HEMA), MMA,terpolymers of HEMA: MMA:MA synthesized in the presence ofN,N′-bis(methacryloyloxyethyloxycarbonylamino)-azobenzene, azopolymers,enteric coated timed release system (Time Clock® from PharmaceuticalProfiles, Ltd., UK) and calcium pectinate can be included in the coat.

The enteric coat can comprise one or more materials that do notdissolve, disintegrate, or erode in the stomach and during the period oftime that the capsule resides in the stomach. A material that easilyadapts to this kind of requirement is a poly(vinylpyrrolidone)-vinylacetate copolymer, such as the material supplied by BASF under itsKollidon VA64 trademark. The enteric coat can also comprise povidone,which is supplied by BASF under its Kollidon K 30 trademark, andhydroxypropyl methylcellulose, which is supplied by Dow under itsMethocel E-15 trademark.

The enteric coat can also comprise other materials suitable which aresubstantially resistant to gastric juices and which will promote eitherenteric or colonic release. Representative materials that keep theirintegrity in the stomach can comprise a member selected from the groupconsisting of (a) keratin, keratin sandarac-tolu, salol (phenylsalicylate), salol beta-naphthylbenzoate and acetotannin, salol withbalsam of Peru, salol with tolu, salol with gum mastic, salol andstearic acid, and salol and shellac; (b) a member selected from thegroup consisting of formalized protein, formalized gelatin, andformalized cross-linked gelatin and exchange resins; (c) a memberselected from the group consisting of myristic acid-hydrogenated castoroil-cholesterol, stearic acid-mutton tallow, stearic acid-balsam oftolu, and stearic acid-castor oil; (d) a member selected from the groupconsisting of shellac, ammoniated shellac, ammoniated shellac-salol,shellac-wool fat, shellac-acetyl alcohol, shellac-stearic acid-balsam oftolu, and shellac n-butyl stearate; (e) a member selected from the groupconsisting of abietic acid, methyl abictate, benzoin, balsam of tolu,sandarac, mastic with tolu, and mastic with tolu, and mastic with acetylalcohol; (f) acrylic resins represented by anionic polymers synthesizedfrom methacrylate acid and methacrylic acid methyl ester, copolymericacrylic resins of methacrylic and methacrylic acid and methacrylic acidalkyl esters, copolymers of alkacrylic acid and alkacrylic acid alkylesters, acrylic resins such asdimethylaminoethylmethacrylate-butylmethacrylate-methylmethacrylatecopolymer of 150,000 molecular weight, methacrylicacid-methylmethacrylate 50:50 coploymer of 135,000 molecular weight,methacrylic acid-methylmethacrylate-30:70-copolymer of 135,000 mol. wt.,methacrylic acid-dimethylaminoethyl-methacrylate-ethylacrylate of750,000 mol. wt., methacrylic acid-methylmethacrylate-ethylacrylate of1,000,000 mol. wt., and ethylacrylate-methylmethacrylate-ethylacrylateof 550,000 mol. wt; and, (g) an enteric composition comprising a memberselected from the group consisting of cellulose acetyl phthalate,cellulose diacetyl phthalate, cellulose triacetyl phthalate, celluloseacetate phthalate, hydroxypropylmethylcellulose phathalate, sodiumcellulose acetate phthalate, cellulose ester phthalate, cellulose etherphthalate, methylcellulose phthalate, cellulose ester-ether phthalate,hydroxypropyl cellulose phthalate, alkali salts of cellulose acetatephthalate, alkaline earth salts of cellulose acetate phthalate, calciumsalt of cellulose acetate phthalate, ammonium salt of hydroxypropylmethylcellulose phthalate, cellulose acetate hexahydrophthalate,hydroxypropyl methylcellulose hexahydrophthalate, polyvinyl acetatephthalate diethyl phthalate, dibutyl phthalate, dialkyl phthalatewherein the alkyl comprises from 1 to 7 straight and branched alkylgroups, aryl phthalates, and other materials known to one or ordinaryskill in the art.

Plasticizers that can be used in the coating(s), e.g. enteric coat orfinish coat, of the capsule include all those that are generallyincorporated into polymeric coatings of drug delivery devices.Plasticizers generally improve the mechanical properties and increasethe flexibility of the polymeric film. Plasticizers generally reducecohesive intermolecular forces and increase mobility of polymer chains,thus reducing polymer-polymer interactions. This action is responsiblefor the changes to the properties of the polymers and films thereof suchas a reduction of Tg (glass transition temperature) or softeningtemperature and the elastic module, increasing polymer flexibility, thusfacilitating the process of formation of the membrane or film. Apreferred pharmaceutical plasticizer is non-toxic and non-irritating;has a reduced tendency to migrate, extrude or volatilize; and has goodmiscibility with the polymer(s) in the film. Plasticizers that can beused in the coating include, for example and without limitation, acetyltriethyl citrate, acetyl tributyl citrate, triethyl citrate, acetylatedmonoglycerides, glycerol, polyethylene glycol, triacetin, propyleneglycol, dibutyl phthalate, diethyl phthalate, isopropyl phthalate,dimethyl phthalate, dactyl phthalate, dibutyl sebacate, dimethylsebacate, castor oil, glycerol monostearate, fractionated coconut oil,poly(ethylene glycol) (PEG), others or a combination thereof. In someembodiments, the plasticizer is PEG having a molecular weight of 200 to8000, ester of citric acid, ester of phthalic acid. Specificplasticizers include PEG having a molecular weight of 200 to 8000,triethyl citrate, tributyl citrate, diethyl phthalate, and dibutylsebacate.

Suitable plasticizers also include, by way of example and withoutlimitation, low molecular weight polymers, oligomers, copolymers, oils,small organic molecules, low molecular weight polyols having aliphatichydroxyls, ester-type plasticizers, glycol esters, poly(propyleneglycol), multi-block polymers, single-block polymers, low molecularweight poly(ethylene glycol), citrate ester-type plasticizers,triacetin, propylene glycol and glycerin. Such plasticizers can alsoinclude ethylene glycol, 1,2-butylene glycol, 2,3-butylene glycol,styrene glycol, diethylene glycol, triethylene glycol, tetraethyleneglycol and other poly(ethylene glycol) compounds, monopropylene glycolmonoisopropyl ether, propylene glycol monoethyl ether, ethylene glycolmonoethyl ether, diethylene glycol monoethyl ether, sorbitol lactate,ethyl lactate, butyl lactate, ethyl glycolate, dibutylsebacate,acetyltributylcitrate, triethyl citrate, acetyl triethyl citrate,tributyl citrate and allyl glycolate. All such plasticizers arecommercially available from sources such as Aldrich or Sigma ChemicalCo. A combination of plasticizers may also be used in the presentformulation. The PEG based plasticizers are commercially available orcan be made by a variety of methods, such as disclosed in Poly (ethyleneglycol) Chemistry: Biotechnical and Biomedical Applications (J. M.Harris, Ed.; Plenum Press, NY) the disclosure of which is herebyincorporated by reference.

As used herein, the term “antioxidant” is intended to mean an agent thatinhibits oxidation and is thus used to prevent the deterioration ofpreparations by the oxidative process. Such compounds include, by way ofexample and without limitation, ascorbic acid, ascorbic palmitate,Vitamin E, Vitamin E derivative, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate,sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate,sodium metalbisulfite and other such materials known to those ofordinary skill in the art.

Although not necessary, the formulation of the present invention mayinclude a chelating agent, preservative, adsorbents, acidifying agent,alkalizing agent, antifoaming agent, buffering agent, colorant,electrolyte, flavorant, polishing agent, salt, stabilizer, sweeteningagent, tonicity modifier, antiadherent, binder, diluent, directcompression excipient, disintegrant, glidant, lubricant, opaquant,polishing agent, plasticizer, other pharmaceutical excipient, or acombination thereof.

As used herein, the term chelating agent is intended to mean a compoundthat chelates metal ions in solution. Exemplary chelating agents includeEDTA (tetrasodium ethylenediaminetetraacetate), DTPA (pentasodiumdiethylenetriaminepentaacetate), HEDTA (trisodium salt ofN-(hydroxyethyl)-ethylenediaminetriacetic acid), NTA (trisodiumnitrilotriacetate), disodium ethanoldiglycine (Na₂EDG), sodiumdiethanolglycine (DEGNa), citric acid, and other compounds known tothose of ordinary skill in the art.

As used herein, the term “adsorbent” is intended to mean an agentcapable of holding other molecules onto its surface by physical orchemical (chemisorption) means. Such compounds include, by way ofexample and without limitation, powdered and activated charcoal andother materials known to one of ordinary skill in the art.

As used herein, the term “alkalizing agent” is intended to mean acompound used to provide an alkaline medium. Such compounds include, byway of example and without limitation, ammonia solution, ammoniumcarbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodiumborate, sodium carbonate, sodium bicarbonate, sodium hydroxide,triethanolamine, and trolamine and others known to those of ordinaryskill in the art.

As used herein, the term “acidifying agent” is intended to mean acompound used to provide an acidic medium. Such compounds include, byway of example and without limitation, acetic acid, amino acid, citricacid, fumaric acid and other alpha hydroxy acids, hydrochloric acid,ascorbic acid, and nitric acid and others known to those of ordinaryskill in the art.

As used herein, the term “antiadherent” is intended to mean an agentthat prevents the sticking of tablet formulation ingredients to punchesand dies in a tableting machine during production. Such compoundsinclude, by way of example and without limitation, magnesium stearate,talc, calcium stearate, glyceryl behenate, polyethylene glycol (PEG),hydrogenated vegetable oil, mineral oil, stearic acid and othermaterials known to one of ordinary skill in the art.

As used herein, the term “binder” is intended to mean a substance usedto cause adhesion of powder particles in granulations. Such compoundsinclude, by way of example and without limitation, acacia, alginic acid,carboxymethylcellulose sodium, poly(vinylpyrrolidone), compressiblesugar (e.g., NuTab), ethylcellulose, gelatin, liquid glucose,methylcellulose, povidone and pregelatinized starch and other materialsknown to one of ordinary skill in the art.

Exemplary binders include acacia, tragacanth, gelatin, starch, cellulosematerials such as methyl cellulose and sodium carboxy methyl cellulose,alginic acids and salts thereof, polyethylene glycol, guar gum,polysaccharide, bentonites, sugars, invert sugars, poloxamers (PLURONIC™F68, PLURONIC™ F127), collagen, albumin, gelatin, cellulosics innonaqueous solvents, combinations thereof and the like. Other bindersinclude, for example, polypropylene glycol,polyoxyethylene-polypropylene copolymer, polyethylene ester,polyethylene sorbitan ester, polyethylene oxide, combinations thereofand other materials known to one of ordinary skill in the art.

As used herein, the term “antifoaming agent” is intended to mean acompound or compounds that prevents or reduces the amount of foamingthat forms on the surface of the fill composition. Suitable antifoamingagents include by way of example and without limitation, dimethicone,SIMETHICONE, octoxynol and others known to those of ordinary skill inthe art.

As used herein, the term “buffering agent” is intended to mean acompound used to resist a change in pH upon dilution or addition of acidor alkali. Such compounds include, by way of example and withoutlimitation, potassium metaphosphate, potassium phosphate, monobasicsodium acetate and sodium citrate anhydrous and dehydrate and other suchmaterials known to those of ordinary skill in the art.

As used herein, the term “diluent” or “filler” is intended to mean inertsubstances used as fillers to create the desired bulk, flow properties,and compression characteristics in the preparation of tablets andcapsules. Such compounds include, by way of example and withoutlimitation, dibasic calcium phosphate, kaolin, lactose, sucrose,mannitol, microcrystalline cellulose, powdered cellulose, precipitatedcalcium carbonate, sorbitol, and starch and other materials known to oneof ordinary skill in the art.

As used herein, the term “direct compression excipient” is intended tomean a compound used in direct compression tablet formulations. Suchcompounds include, by way of example and without limitation, dibasiccalcium phosphate (e.g., Ditab) and other materials known to one ofordinary skill in the art.

As used herein, the term “glidant” is intended to mean an agent used intablet and capsule formulations to promote flowability of thegranulation. Such compounds include, by way of example and withoutlimitation, colloidal silica, cornstarch, talc, calcium silicate,magnesium silicate, colloidal silicon, silicon hydrogel and othermaterials known to one of ordinary skill in the art.

As used herein, the term “lubricant” is intended to mean a substanceused in the instant formulations to reduce friction during compressionor other processing. Such compounds include, by way of example andwithout limitation, calcium stearate, magnesium stearate, mineral oil,stearic acid, and zinc stearate and other materials known to one ofordinary skill in the art.

As used herein, the term “opaquant” is intended to mean a compound usedto render a capsule or a tablet coating opaque. May be used alone or incombination with a colorant. Such compounds include, by way of exampleand without limitation, titanium dioxide, talc and other materials knownto one of ordinary skill in the art.

As used herein, the term “disintegrant” is intended to mean a compoundused in solid dosage forms to promote the disruption of the solid massinto smaller particles that are more readily dispersed or dissolved.Exemplary disintegrants include, by way of example and withoutlimitation, starches such as corn starch, potato starch, pre-gelatinizedand modified starches thereof, sweeteners, clays, such as bentonite,microcrystalline cellulose (e.g., Avicel), carboxymethylcellulosecalcium, cellulose polyacrilin potassium (e.g., Amberlite), alginates,sodium starch glycolate, gums such as agar, guar, locust bean, karaya,pectin, tragacanth; crospovidone and other materials known to one ofordinary skill in the art.

As used herein, the term “preservative” is intended to mean a compoundused to prevent the growth of microorganisms. Such compounds include, byway of example and without limitation, benzalkonium chloride,benzethonium chloride, benzoic acid, benzyl alcohol, cetylpyridiniumchloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuricnitrate, phenylmercuric acetate, thimerosal, metacresol, myristylgammapicolinium chloride, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, sorbic acid, thymol, and methyl, ethyl,propyl, or butyl parabens and others known to those of ordinary skill inthe art.

As used herein, the term “polishing agent” is intended to mean acompound used to impart brightness to the surface of dosage forms. Suchcompounds include, by way of example and without limitation, carnaubawax, white wax, combinations thereof and other such materials known tothose of ordinary skill in the art.

As used herein, the term “colorant” is intended to mean a compound usedto impart color to pharmaceutical preparations. Such compounds include,by way of example and without limitation, FD&C Red No. 3, FD&C Red No.20, FD&C Yellow No. 6, FD&C Blue No. 2, FD&C Green No. 5, FD&C OrangeNo. 5, FD&C Red No. 8, caramel, and iron oxide (black, red, yellow),other FD&C dyes and natural coloring agents such as grape skin extract,beet red powder, beta-carotene, annato, carmine, turmeric, paprika,combinations thereof and other such materials known to those of ordinaryskill in the art.

As used herein, the term “flavorant” is intended to mean a compound usedto impart a pleasant flavor and often odor to a pharmaceuticalpreparation. Exemplary flavoring agents or flavorants include syntheticflavor oils and flavoring aromatics and/or natural oils, extracts fromplants, leaves, flowers, fruits and so forth and combinations thereof.These may also include cinnamon oil, oil of wintergreen, peppermintoils, clove oil, bay oil, anise oil, eucalyptus, thyme oil, cedar leaveoil, oil of nutmeg, oil of sage, oil of bitter almonds and cassia oil.Other useful flavors include vanilla, citrus oil, including lemon,orange, grape, lime and grapefruit, and fruit essences, including apple,pear, peach, strawberry, raspberry, cherry, plum, pineapple, apricot andso forth. Flavors, which have been found to be particularly useful,include commercially available orange, grape, cherry and bubble gumflavors and mixtures thereof. The amount of flavoring may depend on anumber of factors, including the desired organoleptic effect. Flavorswill be present in any amount as desired by the artisan of ordinaryskill in the art. Particularly preferred flavors are the grape andcherry flavors and citrus flavors such as orange.

As used herein, the term “stabilizer” is intended to mean a compoundused to stabilize a active agent against physical, chemical, orbiochemical process that would otherwise reduce the therapeutic activityof the agent. Suitable stabilizers include, by way of example andwithout limitation, albumin, sialic acid, creatinine, glycine and otheramino acids, niacinamide, sodium acetyltryptophonate, zinc oxide,sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethyleneglycols, sodium caprylate and sodium saccharin and others known to thoseof ordinary skill in the art.

As used herein, the term “sweetening agent” is intended to mean acompound used to impart sweetness to a preparation. Such compoundsinclude, by way of example and without limitation, aspartame, dextrose,glycerin, mannitol, saccharin sodium, sorbitol, sucrose, fructose, sugarsubstitute, artificial sweetener, and other such materials known tothose of ordinary skill in the art.

As used herein, the term “tonicity modifier” is intended to mean acompound or compounds that can be used to adjust the tonicity of theliquid formulation. Suitable tonicity modifiers include glycerin,lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol,trehalose and others known to those or ordinary skill in the art.

Plasticizers can also be included to modify the properties andcharacteristics of the polymers used in a pharmaceutical dosage form. Asused herein, the term “plasticizer” includes all compounds capable ofplasticizing or softening a polymer or binder used in invention. Theplasticizer should be able to lower the melting temperature or glasstransition temperature (softening point temperature) of the polymer orbinder. Plasticizers, such as low molecular weight PEG, generallybroaden the average molecular weight of a polymer in which they areincluded thereby lowering its glass transition temperature or softeningpoint. Plasticizers also generally reduce the viscosity of a polymer. Itis possible the plasticizer will impart some particularly advantageousphysical properties to the osmotic device of the invention.

Plasticizers useful in the invention can include, by way of example andwithout limitation, low molecular weight polymers, oligomers,copolymers, oils, small organic molecules, low molecular weight polyolshaving aliphatic hydroxyls, ester-type plasticizers, glycol ethers,poly(propylene glycol), multi-block polymers, single block polymers, lowmolecular weight poly(ethylene glycol), citrate ester-type plasticizers,triacetin, propylene glycol and glycerin. Such plasticizers can alsoinclude ethylene glycol, 1,2-butylene glycol, 2,3-butylene glycol,styrene glycol, diethylene glycol, triethylene glycol, tetraethyleneglycol and other poly(ethylene glycol) compounds, monopropylene glycolmonoisopropyl ether, propylene glycol monoethyl ether, ethylene glycolmonoethyl ether, diethylene glycol monoethyl ether, sorbitol lactate,ethyl lactate, butyl lactate, ethyl glycolate, dibutylsebacate,acetyltributylcitrate, triethyl citrate, acetyl triethyl citrate,tributyl citrate and allyl glycolate. All such plasticizers arecommercially available from sources such as Aldrich or Sigma ChemicalCo. It is also contemplated and within the scope of the invention, thata combination of plasticizers may be used in the present formulation.The PEG based plasticizers are available commercially or can be made bya variety of methods, such as disclosed in Poly(ethylene glycol)Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Ed.;Plenum Press, NY) the disclosure of which is hereby incorporated byreference.

The composition of the invention can be included in any dosage form.Particular dosage forms include a solid or liquid dosage forms.Exemplary suitable dosage forms include tablet, capsule, pill, caplet,troche, sache, and other such dosage forms known to the artisan ofordinary skill in the pharmaceutical sciences.

Examples 3 and 6 describe an exemplary capsule dosage form. Example 13describes an exemplary tablet dosage form.

The composition of the invention can also include oils such as fixedoils, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil;fatty acids such as oleic acid, stearic acid and isostearic acid; andfatty acid esters such as ethyl oleate, isopropyl myristate, fatty acidglycerides and acetylated fatty acid glycerides. The composition canalso include alcohol such as ethanol, isopropanol, hexadecyl alcohol,glycerol and propylene glycol; glycerol ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol; ethers such as poly(ethyleneglycol) 450; petroleum hydrocarbons such as mineral oil and petrolatum;water; a pharmaceutically suitable surfactant, suspending agent oremulsifying agent; or mixtures thereof.

It should be understood that the compounds used in the art ofpharmaceutical formulation generally serve a variety of functions orpurposes. Thus, if a compound named herein is mentioned only once or isused to define more than one term herein, its purpose or function shouldnot be construed as being limited solely to that named purpose(s) orfunction(s).

As used herein, the term “oleandrin” is taken to mean all known forms ofoleandrin unless otherwise specified. Oleandrin can be present inracemic, optically pure or optically enriched form. Nerium oleanderplant material can be obtained from commercial plant suppliers such asAldridge Nursery, Atascosa, Tex.

One or more of the components of the formulation can be present in itsfree base or pharmaceutically acceptable salt form. As used herein,“pharmaceutically acceptable salt” refers to a compound that has beenmodified by reacting it with an acid as needed to form an ionicallybound pair. Examples of pharmaceutically acceptable salts includeconventional non-toxic salts formed, for example, from non-toxicinorganic or organic acids. Suitable non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfonic, sulfamic, phosphoric, nitric and others known tothose of ordinary skill in the art. The salts prepared from organicacids such as amino acids, acetic, propionic, succinic, glycolic,stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and others known to those of ordinaryskill in the art. Lists of other suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th). ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, the relevant disclosure of which is herebyincorporated by reference.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith tissues of human beings and animals and without excessive toxicity,irritation, allergic response, or any other problem or complication,commensurate with a reasonable benefit/risk ratio.

The amount of oleandrin incorporated in a unit dose of the inventionwill be at least one or more dosage forms and can be selected accordingto known principles of pharmacy. An effective amount of therapeuticcompound is specifically contemplated. By the term “effective amount”,it is understood that, with respect to, for example, pharmaceuticals, apharmaceutically effective amount is contemplated. A pharmaceuticallyeffective amount is the amount or quantity of tramadol which is enoughfor the required or desired therapeutic response, or in other words, theamount, which is sufficient to elicit an appreciable biological responsewhen, administered to a patient. The appreciable biological response mayoccur as a result of administration of single or multiple unit doses ofan active substance. A unit dose may comprise one or more dosage forms,such as capsules. It will be understood that the specific dose level forany patient will depend upon a variety of factors including theindication being treated, severity of the indication, patient health,age, sex, weight, diet, pharmacological response, the specific dosageform employed and other such factors.

The dose of SCF extract will depend upon the concentration oftherapeutically effective components therein. The in vitro cellularassays disclosed herein can be used to determine the relative potency ofthe extract. The desired dose for oral administration is up to 5 dosageforms although as few as one and as many as ten dosage forms may beadministered. Exemplary dosage forms contain 38.5 mg of the SCF extractper dosage form, for a total 38.5 to 385 mg of extract or of cardiacglycoside (1 to 10 dosage forms) per dose.

The oleandrin is present in the dosage form in an amount sufficient toprovide a subject with an initial dose of oleandrin of 0.5 to 5 mg. Someembodiments of the dosage form are not enteric coated and release theircharge of oleandrin within a period of 0.5 to 1 hours or less. Someembodiments of the dosage form are enteric coated and release theircharge of oleandrin downstream of the stomach, such as from the jejunum,ileum, small intestine, and/or large intestine (colon). Oleandrin fromenterically coated dosage forms will be released into systemiccirculation within 2-3 hr after oral administration. Based onpreliminary animal dosing data it is anticipated that 50 to 75% of anadministered dose of oleander extract will be orally bioavailabletherefore providing 0.25 to 0.4 mg oleandrin per dosage form. Given anaverage blood volume in adult humans of 5 liters, the anticipatedoleandrin plasma concentration will be in the range of 0.05 to 2 ug/ml.

The recommended daily dose of oleandrin, present in the SCF extract, isgenerally about 0.9 to 5 mg twice daily or about every 12 hours, with amaximum dose of about 1.8 to 10 mg/day.

If desired, the dosage form of the invention can be coated with a finishcoating as is commonly done in the art to provide the desired shine,color, taste or other aesthetic characteristics. Materials suitable forpreparing the finish coating are well known to those of ordinary skillin the art.

The in vitro release profile of an enteric coated capsule (coatedaccording to Example 6) containing Formulation #2 or Formulation #3 wasevaluated according to the USP dissolution method for enteric coateddosage forms. The dissolution profile is depicted in FIG. 2. The USPpaddle method was used for the dissolution testing with the paddle speedset at 50 rpm. In the first two hours, 750 mL of 0.1 N hydrochloric acidsolution was used as the dissolution medium. After 2 hours, 250 mMsodium phosphate solution was added into 750 mL 0.1 N hydrochloric acidsolution to adjust pH to 6.8. The results indicate that less than 5%drug was released in the acid stage and greater than 75% of drug wasreleased within one hour following the adjustment in the dissolutionmedium

In view of the above description and the examples below, one of ordinaryskill in the art will be able to practice the invention as claimedwithout undue experimentation. The foregoing will be better understoodwith reference to the following examples that detail certain proceduresfor the preparation of embodiments of the present invention. Allreferences made to these examples are for the purposes of illustration.The following examples should not be considered exhaustive, but merelyillustrative of only a few of the many embodiments contemplated by thepresent invention.

EXAMPLE 1 Supercritical Fluid Extraction of Powdered Oleander LeavesMethod A. With Carbon Dioxide.

Powdered oleander leaves were prepared by harvesting, washing, anddrying oleander leaf material, then passing the oleander leaf materialthrough a comminuting and dehydrating apparatus such as those describedin U.S. Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. Theweight of the starting material used was 3.94 kg.

The starting material was combined with pure CO₂ at a pressure of 300bar (30 MPa, 4351 psi) and a temperature of 50° C. (122° F.) in anextractor device. A total of 197 kg of CO₂ was used, to give a solventto raw material ratio of 50:1. The mixture of CO₂ and raw material wasthen passed through a separator device, which changed the pressure andtemperature of the mixture and separated the extract from the carbondioxide.

The extract (65 g) was obtained as a brownish, sticky, viscous materialhaving a nice fragrance. The color was likely caused by chlorophyll andother residual chromophoric compounds. For an exact yield determination,the tubes and separator were rinsed out with acetone and the acetone wasevaporated to give an addition 9 g of extract. The total extract amountwas 74 g. Based on the weight of the starting material, the yield of theextract was 1.88%. The content of oleandrin in the extract wascalculated using high pressure liquid chromatography and massspectrometry to be 560.1 mg, or a yield of 0.76%.

Method B. With Mixture of Carbon Dioxide and Ethanol

Powdered oleander leaves were prepared by harvesting, washing, anddrying oleander leaf material, then passing the oleander leaf materialthrough a comminuting and dehydrating apparatus such as those describedin U.S. Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. Theweight of the starting material used was 3.85 kg.

The starting material was combined with pure CO₂ and 5% ethanol as amodifier at a pressure of 280 bar (28 MPa, 4061 psi) and a temperatureof 50° C. (122° F.) in an extractor device. A total of 160 kg of CO₂ and8 kg ethanol was used, to give a solvent to raw material ratio of 43.6to 1. The mixture of CO₂, ethanol, and raw material was then passedthrough a separator device, which changed the pressure and temperatureof the mixture and separated the extract from the carbon dioxide.

The extract (207 g) was obtained after the removal of ethanol as a darkgreen, sticky, viscous mass obviously containing some chlorophyll. Basedon the weight of the starting material, the yield of the extract was5.38%. The content of oleandrin in the extract was calculated using highpressure liquid chromatography and mass spectrometry to be 1.89 g, or ayield of 0.91%.

EXAMPLE 2 Hot-Water Extraction of Powdered Oleander Leaves ComparativeExample

Hot water extraction is typically used to extract oleandrin and otheractive components from oleander leaves. Examples of hot water extractionprocesses can be found in U.S. Pat. Nos. 5,135,745 and 5,869,060.

A hot water extraction was carried out using 5 g of powdered oleanderleaves. Ten volumes of boiling water (by weight of the oleander startingmaterial) were added to the powdered oleander leaves and the mixture wasstirred constantly for 6 hours. The mixture was then filtered and theleaf residue was collected and extracted again under the sameconditions. The filtrates were combined and lyophilized. The appearanceof the extract was brown. The dried extract material weighed about 1.44g. 34.21 mg of the extract material was dissolved in water and subjectedto oleandrin content analysis using high pressure liquid chromatographyand mass spectrometry. The amount of oleandrin was determined to be 3.68mg. The oleandrin yield, based on the amount of extract, was calculatedto be 0.26%. The Table 1 below shows a comparison between the oleandrinyields for the two supercritical carbon dioxide extractions of Example 1and the hot water extraction.

TABLE 1 Comparison of Yields Oleandrin yield based Extraction Medium ontotal extract weight Supercritical Carbon Dioxide: Example 1, 0.76%Method A Supercritical Carbon Dioxide: Example 1, 0.91% Method B HotWater Extraction: Example 2 0.26%

EXAMPLE 3 Preparation of Pharmaceutical Compositions

In each of the following methods, the SCF extract contained about 25 mgof oleandrin per gram of extract.

Method A. Cremophor-Based Drug Delivery System

The following ingredients were provided in the amounts indicated.

Reagent Percent of Formulation Name Function (% w/w) SCF extract Activeagent 3.7 Vitamin E Antioxidant 0.1 Labrasol Surfactant 9.2 EthanolCo-solvent 9.6 Cremophor EL Surfactant 62.6 Cremophor RH40 Surfactant14.7

The excipients were dispensed into a jar and shook in a New BrunswickScientific C24KC Refrigerated Incubator shaker for 24 hours at 60° C. toensure homogeneity. The samples were then pulled and visually inspectedfor solubilization. Both the API and remainder of the extract weretotally dissolved for all formulations after 24 hours.

Method B. GMO/Cremophor-Based Drug Delivery System

The following ingredients were provided in the amounts indicated.

Reagent Percent of Formulation Name Function (% w/w) SCF extract Activeagent 4.7 Vitamin E Antioxidant 0.1 Labrasol Surfactant 8.5 EthanolCo-solvent 7.6 Cremophor EL Surfactant 56.1 Glycerol MonooleateSurfactant 23.2

The procedure of Method A was followed.

Method C. Labrasol-Based Drug Delivery System

The following ingredients were provided in the amounts indicated.

Reagent Percent of Formulation Name Function (% w/w) SCF extract Activeagent 3.7 Vitamin E Antioxidant 0.1 Labrasol Surfactant 86.6 EthanolCo-solvent 9.6

The procedure of Method A was followed.

Method D. Vitamin E-TPGS Based Micelle Forming System

The following ingredients were provided in the amounts indicated.

Component Function Weight % (w/w) Vitamin E Antioxidant 1.0 Vitamin ETPGS Surfactant 95.2 SCF extract Active agent 3.8

The procedure of Method A was followed.

Method E. Multi-Component Drug Delivery System

The following ingredients were provided in the amounts indicated.

Component Weight (g) Weight % (w/w) Vitamin E 10.0 1.0 Cremophor ELP580.4 55.9 Labrasol 89.0 8.6 Glycerol 241.0 23.2 Monooleate Ethanol 80.07.7 SCF extract 38.5 3.7 Total 1038.9 100

The procedure of Method A was followed.

EXAMPLE 4 In Vitro Dissolution Assay Method A. Screening Studies toIdentify Materials Suitable for the Solubilizer

A screening assay was conducted to determine which materials might besuitable for use in the liquid composition. Preliminary solubilitystudies were performed by preparing binary mixtures containing anexcipient and the SCF extract. A suitable single excipient solubilizes amajor portion of the oleandrin and other components present in theextract.

The SCF extract was placed in the solubilizer at a concentration of 77mg SCF extract per mL of excipient in 20 mL scintillation vials. Afterweighing out the solubilizer and extract in the vials, the samples weremixed using a vortex mixer. Those samples that did not go into solutionafter being vortexed at ambient conditions were heated in a hot waterbath at 100° C. for 15 minutes, vortexed, sonicated for 10 minutes, andthen reheated for another 15 minutes at 100° C. The samples were thencooled to ambient condition for 24 hours and visually inspected for thepresence of particles.

Exemplary suitable water soluble solvents included: ethanol, Lauroglycol90, Pharmasolve, Soluphor P and Triacetin.

Exemplary water insoluble solvent included: Captex 350, Captex 355,glyceryl monooleate, Miglyol 810, olive oil, sesame oil, and Softisan645,

Exemplary surfactants included: Cremophor EL, Cremophor RH40, Gelucire33/01, Gelucire 43/01, Gelucire 44/14, Gelucire 50/13, labrafil M 1944,labrafil M 2125, labrasol, lutrol L44 NF, plurol oleique, span 20, span80 and Tween 80.

Method B. Screening Studies to Identify Solubilizer Suitable for Use inthe Liquid Composition

A screening assay was conducted to determine which materials might besuitable for use as a solubilizer in the liquid composition. A suitablesolubilizer was able to dissolve the SCF extract to make a clear liquidcomposition.

Method C. Dissolution Assay to Evaluate Performance of Solubilizer

An aliquot (one to a few drops) of liquid composition containing SCFextract and solubilizer was placed in 200 ml of phosphate buffer (pH6.8, 50 mM) with stirring at ambient temperature. The clarity of thesolution was then determined.

EXAMPLE 5 Determination of Clarity

Method A. Visual Inspection with the Unaided Eye

A vial containing the sample being analyzed was held up to a lightsource. The presence of suspended solids was determined visually.

Method B. Visual Inspection with a Microscope

An aliquot of liquid composition was placed on a microscope slide andviewed under 1000× magnification. The presence of suspended solids wasdetermined visually.

EXAMPLE 6 Preparation of Enteric Coated Capsules Step I: Preparation ofLiquid-Filled Capsule

Hard gelatin capsules (50 counts, 00 size) were filled with a liquidcomposition of Example 3. These capsules were manually filled with 800mg of the formulation and then sealed by hand with a 50% ethanol/50%water solution. The capsules were then banded by hand with 22% gelatinsolution containing the following ingredients in the amounts indicated.

Ingredient Wt. (g) Gelatin 140.0 Polysorbate 80 6.0 Water 454.0 Total650.0

The gelatin solution mixed thoroughly and allowed to swell for 1-2hours. After the swelling period, the solution was covered tightly andplaced in a 55° C. oven and allowed to liquefy. Once the entire gelatinsolution was liquid, the banding was performed

Using a pointed round 3/0 artist brush, the gelatin solution was paintedonto the capsules. Banding kit provided by Shionogi was used. After thebanding, the capsules were kept at ambient conditions for 12 hours toallow the band to cure.

Step II: Coating of Liquid-Filled Capsule

A coating dispersion was prepared from the ingredients listed in thetable below.

Ingredient Wt. % Solids % Solids (g) g/Batch Eudragit L30D55 40.4 60.576.5 254.9 TEC 1.8 9.0 11.4 11.4 AlTalc 500V 6.1 30.5 38.5 38.5 Water51.7 na na 326.2 Total 100.0 100.0 126.4 631.0

If banded capsules according to Step I were used, the dispersion wasapplied to the capsules to a 20.0 mg/cm² coating level. The followingconditions were used to coat the capsules.

Parameters Set-up Coating Equipment Vector LDCS-3 Batch Size 500 g InletAir Temp. 40° C. Exhaust Air Temp. 27-30° C. Inlet Air Volume 20-25 CFMPan Speed 20 rpm Pump Speed 9 rpm (3.5 to 4.0 g/min) Nozzle Pressure 15psi Nozzle diameter 1.0 mm Distance from tablet bed* 2-3 in *Spraynozzle was set such that both the nozzle and spray path were under theflow path of inlet air.

EXAMPLE 7 Treatment of Skin Related Diseases Such as Cancers IncludingBut not Limited to Prevention of Treatment of Melanoma, Basal CellCarcinoma, and Squamous Cell Carcinoma as Well as NoncancerousInflammatory Skin Diseases Including But not Limited to ActinicKeratosis, Psoriasis, and Eczema

The SCF extract is administered to a subject suffering from malignant ornonmalignant proliferative skin diseases such as those cited above. TheSCF extract is administered as a cream or ointment or contained within adermal patch containing 0.01 mg to 10 mg of SCF extract per unit dose.The subject is administered a unit dose up to three times daily for aperiod of 1 to 14 days or until the skin diseases is in remission. It isexpected that such treatment will significantly lessen or eliminate theinflammation and malignant processes leading to a progression of thedisease. The subject should experience a reduction in the severity ofthe dermal lesion(s) and the eventual resolution of the dermatologicdisease itself. Malignant diseases should be expected to be reduced inrate of growth or inhibited from increase in severity of the disease.Actual regression of established malignant lesions may be expected.

EXAMPLE 8 Prevention of Skin Related Diseases Such as Skin Cancers

The SCF extract is administered to a subject suffering from apredisposition to formation of skin cancer such as those frequentlyexposed to ultraviolet light (from sunlight) or carcinogens fromchemicals. The SCF extract is administered as a cream or ointment orcontained within a dermal patch containing 0.01 to 10 mg of SCF extractper unit dose. The subject is administered a unit dose up to three timesdaily every time exposure to a carcinogen promoting event is anticipated(exposure to sunlight). Such administration could, for example, be madeas a sunscreen for blocking sunlight UV exposure and SCF extract forprevention of tumor induction in dermal tissue. It would be expectedthat such a use of the SCE in a dermal product would block formationand/or promotion of malignant skin disease or nonmalignant skindisorders where proliferation leads to a worsening of the diseaseprocess (e.g. acktinic keratosis, psoriasis and/or eczema).

EXAMPLE 9 Treatment of Solid Tumors in Humans or Other VertebrateAnimals

SCF extract can be used to treat cancers of the rectum, anus, colorectaltissues, head and neck tissues, esophageal tissue, lung (both non smallcell and small cell carcinomas), breast, stomach, pancreas, prostate,liver, kidney, bladder, ureter, ovarian tissue, carcinoid tumors,sarcomas of bone, mesothelioma, and neoplasms of the central nervoussystem.

The SCF extract is administered to a subject suffering from solidmalignant diseases such as those mentioned above. The SCF extract isadministered as an oral dosage form containing 1 to 50 mg of SCF extractper unit dose. The subject is administered a unit dose up to twice dailytimes daily for a period of 28 days/cycle of treatment. Up to threecycles of treatment may be required. The subject should experience tumorgrowth to either slow in rate of proliferation or to regress. Completionresolution of the tumor may occur. The therapy with SCF extract may beused as a sole agent or combined with cytotoxic chemotherapy orradiation treatment or may be combined with appropriate immunotherapywithout causing undue interference with the desired antitumor effect ofconventional therapy.

EXAMPLE 10 Comparison of Cytotoxicity of Hot Water Extract of Neriumoleander to an SCF Extract Made Using Supercritical CO₂ in Two HumanTumor Cell Lines

The cytotoxic potential of both extracts are compared directly with thatof oleandrin. The samples contained the same amounts of oleandrin eventhough their concentration of oleandrin differed due to theconcentration of oleandrin present in the extracts.

BRO (human melanoma) and Panc-1 (human pancreatic cancer) cells(8×10³/well) were plated in a 96 well plate and allowed to attachovernight. Drug or extracts were then added to the cells. After 72 hr ofincubation, relative cell proliferation (relative to control untreatedcells) was assessed by crystal violet staining method.

EXAMPLE 11 HPLC Analysis of Solutions Containing Oleandrin

Samples (oleandrin standard, SCF extract and hot-water extract) wereanalyzed on HPLC (Waters) using the following conditions: Symmetry C18column (5.0 μm, 150×4.6 mm I.D.; Waters); Mobile phase ofMeOH:water=54:46 (v/v) and flow rate at 1.0 ml/min. Detection wavelengthwas set at 217 nm. The samples were prepared by dissolving the compoundor extract in a fixed amount of HPLC solvent to achieve an approximatetarget concentration of oleandrin.

EXAMPLE 12 Evaluation of Anti-Viral Activity of an SCF Extract

The test consists of determining the relative ability of oleanderextract or a positive control (AZT) to inhibit proliferation of the ROJOstrain of HIV-1 in human peripheral blood mononuclear cells (PBMCs).Infected cells are exposed to the drug or extract for 48 hr. The test isused to determine the IC50 of oleander extract (that concentration ofextract producing a 50% inhibition of viral proliferation) versus thatconcentration of extract capable of killing the human PBMC. This is, ineffect, a determination of the therapeutic index of the extract. This isessentially a determination of whether or not the extract can kill HIV-1without killing the PBMC cell itself.

One should observe an IC50 against viral proliferation of about 5.0ug/ml or less while the concentration required to kill cells should nothave been reached even at concentrations as high as 100 ug/ml. The dataobtained suggest that oleander extract should be useful in terms ofinhibiting HIV-1 viral proliferation harbored within PBMC cells.

EXAMPLE 13 Preparation of a Tablet Comprising SCF Extract

An initial tabletting mixture of 3% Syloid 244FP and 97%microcrystalline cellulose (MCC) was mixed. Then, an existing batch ofcomposition prepared according to Example 3 was incorporated into theSyloid/MCC mixture via wet granulation. This mixture is labeled “InitialTabletting Mixture) in the table below. Additional MCC was addedextra-granularly to increase compressibility. This addition to theInitial Tabletting Mixture was labeled as “Extra-granular Addition.” Theresultant mixture from the extra-granular addition was the samecomposition as the “Final Tabletting Mixture.”

Component Weight (g) Weight % (w/w) Initial Tabletting MixtureMicrocrystalline cellulose 48.5 74.2 Colloidal Silicon Dioxide/Syloid1.5 2.3 244FP Formulation from Ex. 3 15.351 23.5 Total 65.351 100.0

Extragranular addition Component Weight (g) Weight % (w/w) InitialTabulating Mixture 2.5 50.0 Microcrystalline cellulose 2.5 50.0 Total 5100.0

Final Tabletting Mixture:

Abbreviated Component Weight (g) Weight % (w/w) Microcrystallinecellulose 4.36 87.11 Colloidal Silicon Dioxide/Syloid 0.06 1.15 244FPFormulation from Ex. 3 0.59 11.75 Total 5.00 100

Final Tabletting Mixture:

Detailed Component Weight (g) Weight % (w/w) Microcrystalline cellulose4.36 87.11 Colloidal Silicon Dioxide/Syloid 0.06 1.15 244FP Vitamin E0.01 0.11 Cremophor ELP 0.33 6.56 Labrasol 0.05 1.01 Glycerol Monooleate0.14 2.72 Ethanol 0.05 0.90 SCF extract 0.02 0.44 Total 5.00 100.00

Syloid 244FP is a colloidal silicon dioxide manufactured by GraceDavison. Colloidal silicon dioxide is commonly used to provide severalfunctions, such as an adsorbant, glidant, and tablet disintegrant.Syloid 244FP was chosen for its ability to adsorb 3 times its weight inoil and for its 5.5 micron particle size.

EXAMPLE 14 HPLC Carbohydrate Analysis

Carbohydrate content in oleander extracts was determined using themethod of Yemm and Willis (The Estimation of Carbohydrates in PlantExtracts by Anthrone, 1954, 57:508-514). Briefly, aliquots of plantextracts were added to a tube containing anthrone solution (0.2 ganthrone dissolved in 100 ml H₂SO₄) and chilled on ice. The resultingcolor was read in a spectrophotometer at 625 nm using a 1 cm cell. Astandard curve consisting of serial dilutions of glucose was used toquantify the results. Other sugars such as fructose, arabinose andrhamnose also react with the anthrone solution and thus this method is ameasure of many carbohydrates in the plant extract.

The above is a detailed description of particular embodiments of theinvention. It will be appreciated that, although specific embodiments ofthe invention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims. All of the embodiments disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure.

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 20. Asupercritical fluid extract comprising oleandrin.
 21. The supercriticalfluid extract of claim 20, wherein the extract has been prepared bysupercritical fluid extraction of oleandrin-containing plant mass. 22.The supercritical fluid extract of claim 21, wherein the supercriticalfluid extraction has been conducted with a supercritical fluidcomprising carbon dioxide.
 23. The supercritical fluid extract of claim22, wherein the supercritical fluid further comprises a modifier. 24.The supercritical fluid extract of claim 21, wherein theoleandrin-containing plant mass is Nerium species or Thevetia species.25. The supercritical fluid extract of claim 24, wherein the Neriumspecies is Nerium oleander.
 26. The supercritical fluid extract of claim24, wherein the Thevetia species is Thevetia nerifolia.
 27. Asupercritical fluid extract comprising cardiac glycoside.
 28. Thesupercritical fluid extract of claim 27, wherein the extract has beenprepared by supercritical fluid extraction of cardiacglycoside-containing plant mass.
 29. The supercritical fluid extract ofclaim 28, wherein the supercritical fluid extraction has been conductedwith a supercritical fluid comprising carbon dioxide.
 30. Thesupercritical fluid extract of claim 29, wherein the supercritical fluidfurther comprises a modifier.
 31. The supercritical fluid extract ofclaim 27 further comprising at least one other supercritical fluidextractable pharmacologically active agent obtained by way of thesupercritical fluid extraction.
 32. The supercritical fluid extract ofclaim 31, wherein the at least one other supercritical fluid extractablepharmacologically active agent contributes to the therapeutic efficacyof the cardiac glycoside when the extract is administered to a subject.33. The supercritical fluid extract of claim 32, wherein the at leastone other supercritical fluid extractable pharmacologically active agentfunctions additively or synergistically to contribute to the therapeuticefficacy of the cardiac glycoside when the extract is administered to asubject.
 34. A pharmaceutical dosage form comprising a supercriticalfluid extract of according to claim
 20. 35. A method of treating adisease or disorder therapeutically responsive to cardiac glycosidetherapy in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of asupercritical fluid extract according to claim
 27. 36. The method ofclaim 38, wherein the extent of Akt phosphorylation in the cancer cellsis inhibited or reduced by said treating.
 37. The method of claim 38,wherein the expression of pERK in the cancer cells is enhanced by saidtreating.
 38. A method of inhibiting the proliferation of cancer cellscomprising treating the cancer cells with an effective of amountsupercritical fluid extract according to claim
 20. 39. The method ofclaim 35, wherein the supercritical fluid extract is of a cardiacglycoside-containing plant mass.
 40. The method of claim 39, wherein thecardiac glycoside comprises oleandrin.
 41. The method of claim 40,wherein the supercritical fluid extract further comprises at least oneother pharmacologically active agent.
 42. The method of claim 41,wherein the at least one other pharmacologically active agentcontributes to the therapeutic efficacy of the cardiac glycoside whenthe extract is administered to a subject.
 43. The method of claim 42,wherein the at least one other pharmacologically active agent functionsadditively or synergistically to contribute to the therapeutic efficacyof the cardiac glycoside when the extract is administered to a subject.44. The method of claim of claim 39, wherein the oleandrin-containingplant mass is Nerium species or Thevetia species.
 45. The method ofclaim 44, wherein the Nerium species is Nerium oleander.
 46. The methodof claim 44, wherein the Thevetia species is Thevetia nerifolia.
 47. Themethod of claim 44, wherein the cardiac glycoside comprises oleandrin.48. The method of claim 47, wherein the supercritical fluid extractfurther comprises at least one other supercritical fluid extractablepharmacologically active agent.
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 60. Thesupercritical fluid extract of claim 20, wherein the SCF extractcomprises oleandrin, oleandrigenin, ursolic acid, betulinic acid,odoroside, neritaloside, oleanolic acid and one or more triterpenes. 61.The supercritical fluid extract of claim 60, wherein the SCF extractcomprises less than 0.01% by weight polysaccharide.
 62. Thesupercritical fluid extract of claim 60, wherein the SCF extract furthercomprises one or more compounds selected from the group consisting ofneritaloside; oleanolic acid; ursolic acid; betulinic acid; betulin(urs-12-ene-3β,28-diol); 28-norurs-12-en-3β-ol; urs-12-en-3β-ol;3β,3β-hydroxy-12-oleanen-28-oic acid; 3β,20α-dihydroxyurs-21-en-38-oicacid; 3β,27-dihydroxy-12-ursen-38-oic acid;3β,13β-dihydroxyurs-11-en-28-oic acid;3β,12α-dihydroxyoleanan-28,13β-olide; and3β,27-dihydroxy-12-oleanan-28-oic acid.
 63. The supercritical fluidextract of claim 27, wherein the SCF extract comprises one or morecardiac glycosides and one or more cardiac glycoside precursors.
 64. Thesupercritical fluid extract of claim 63, wherein the one or more cardiacglycoside precursors is selected from the group consisting of anaglycone constituent of a cardiac glycoside and a glycone constituent ofa cardiac glycoside.
 65. The supercritical fluid extract of claim 64,wherein: the aglycone is selected from the group consisting ofoleandrigenin, digitoxin, acetyl digitoxins, digitoxigenin, digoxin,acetyl digoxins, digoxigenin, medigoxin, strophanthins, cymarine,ouabain, strophanthidin; and the glycone is selected from the groupconsisting of glucoside, fructoside, and glucuronide.
 66. Thesupercritical fluid extract of claim 65 further comprising one or moreof neritaloside; oleanolic acid; ursolic acid; betulinic acid; betulin(urs-12-ene-3β,28-diol); 28-norurs-12-en-3β-ol; urs-12-en-3β-ol;3β,3β-hydroxy-12-oleanen-28-oic acid; 3β,20α-dihydroxyurs-21-en-38-oicacid; 3β,27-dihydroxy-12-ursen-38-oic acid;3β,13β-dihydroxyurs-11-en-28-oic acid;3β,12α-dihydroxyoleanan-28,13β-olide; and3β,27-dihydroxy-12-oleanan-28-oic acid.
 67. The supercritical fluidextract of claim 63, wherein the SCF extract comprises one or morecardiac glycosides selected from the group consisting of Apocannoside,cymarin, Calotropin, 16α-acetoxycalotropin, 15β-hydroxycalotropin,calactin, 15β-hydroxycalactin, asclepin, 16α-hydroxyasclepin,uscharidin, uscharin, uzarigenin, Digitoxigenin, oleandrigenin,digitoxigenin, α-L-cymaroside, digitoxigeninβ-gentiobiosyl-α-L-cymaroside, Δ¹⁶-digitoxigeninβ-D-glucosyl-α-L-cymaroside, Calotropin, calactin, uscharin,voruscharin, 2″-oxovoruscharin, 2′-O-Acetyl cerleaside A,17α-neriifolin, 17β-neriifolin, cerberin, Hyrcanoside,Securigenin-3β-O-β-6-deoxyguloside,19-hydroxy-sarmentogenin-3β-O-β-6-deoxyguloside,sarmentogenin-3β-O-[α-allosyl-(1→4)-β-6-deoxyalloside],securigenin-3β-O-[α-allosyl-(1→4)-β-6-deoxyalloside], Digoxin,digitoxin, gitoxin, Elaeodendrosides, Acovenosigenin A3-O-α-L-ramnopyranoside, euonymoside A, euonymusoside A, Euonymoside A,Maquiroside A, Oleander, oleandrin, cardenolide N-1, cardenolide N-4,3β-O—(β-D-sarmentosyl)-16β-acetoxy-14-hydroxy-5β,14β-card-20-(22)-enolide,16β-acetoxy-3β,14-dihydroxy-5β,14β-card-20-(22)-enolide,17-epi-11α-hydroxy-6,7-dehydrostrophanthidin-3-O-β-boivinopyranoside,6,7-dehydrostrophanthidin-3-O-β-boivinopyranoside,6,7-dehydrostrophanthidin-3-O-β-oleandropyranoside, Convallatoxin,3′-O-β-D-glucopyranosylcalactin, 12-dehydroxyghalakinoside,6′-dehydroxyghala-kinoside, ghalakinoside, calactin, Periplocin isomers,Rhodexin A, 3-O-β-D-fucopyranosylstrophanthidin,3-O-β-D-quinovopyranosylperiplogenin,3-O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosylcannogenin,3-O-β-D-xylopyranosylperiplogenin,3-O-β-D-quinovopyranosylstrophanthidin,3-O-β-D-xylopyranosylstrophanthidin, 3-O-β-D-fucopyranosylperiplogenin,3-O-α-L-rhamnopyranosylcannogenol, convallatoxin,3-O-α-L-rhamnpyranosylacovenosigenin A, Stebloside, mansonin,Periplogenin digitoxoside, Periplocymarin, digitoxigenin3-O—[O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→4)-β-D-digitoxopyranoside,echujin, corchorusoside C, 3-O-(β-glucopyranosyl)acovenosigenin A,Ouabain, Neriifolin, 3′-O-methylevomonoside, 2′acetylneriifolin,Thevetin A and B, thevetoside, Proscillaridin A, and scillaren A. 68.The supercritical fluid extract of claim 63, wherein the SCF extract isobtained from plant mass selected from the group consisting of: Apocynumcannabinum L. (Apocynaceae), Asclepias curassayica L. (Asclepiadaceae),Beaumontia brevituba Oliver (Apocynaceae), Calotropis procera (Ait.) R.Br. (Asclepiadaceae), Cerbera odollam Gaertner (Apocynaceae), Coronillavaria L. (Fabaceae), Crossopetalum gaumeri (Loes.) Lundell(Celastraceae), Digitalis purpurea L. (Scrophulariaceae), Digitalislanata (Scrophulariaceae), Elaeodendron sp., Euonymus alata (Thunb.)Sieb. (Celastraceae), Euonymus sieboldianus Blume (Celastraceae),Maquira calophylla (P.&E.) C.C. Berg (Moraceae), Nerium oleander L.(Apocynaceae), Nierembergia aristata D. Don (Solanaceae), Ornithogalumumbellatum L. (Hyacinthaceae), Pergularia tomentosa L. (Asclepiadaceae),Periploca graeca L. (Asclepiadaceae), Rhodea japonica (Thunb.) Roth.(Liliaceae), Saussurea stella Maxim. (Asteraceae), Streblus asper Lour.(Moraceae), Streptocaulon juventas (Lour.) Merr. (Asclepiadaceae),Streptocaulon griffithii Hook.f. (Asclepiadaceae), Strophanthus,Thevetia ahouia (L.) A. DC. (Apocynaceae), Thevetia peruviana (Pers.) K.Schum. (Apocynaceae), and Urginea maritime (L.) Baker (Liliaceae).
 69. Apharmaceutical dosage form comprising a supercritical fluid extract ofaccording to claim 27.